High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

PubMed

Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

2015-06-17

High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection. Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.

LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation.

PubMed

Kim, Shin-Hee; Xiao, Sa; Collins, Peter L; Samal, Siba K

2016-06-01

The cleavage site sequence of the fusion (F) protein contributes to a wide range of virulence of Newcastle disease virus (NDV). In this study, we identified other important amino acid sequences of the F protein that affect cleavage and modulation of fusion. We generated chimeric Beaudette C (BC) viruses containing the cleavage site sequence of avirulent strain LaSota (Las-Fc) together with various regions of the F protein of another virulent strain AKO. We found that the F1 subunit is important for cleavage inhibition. Further dissection of the F1 subunit showed that replacement of four amino acids in the BC/Las-Fc protein with their AKO counterparts (T341S, M384I, T385A and I386L) resulted in an increase in fusion and replication in vitro. In contrast, the mutation N403D greatly reduced cleavage and viral replication, and affected protein conformation. These findings will be useful in developing improved live NDV vaccines and vaccine vectors.

Identification of Proteolytic Cleavage Sites of EphA2 by Membrane Type 1 Matrix Metalloproteinase on the Surface of Cancer Cells.

PubMed

Kikuchi, Keiji; Kozuka-Hata, Hiroko; Oyama, Masaaki; Seiki, Motoharu; Koshikawa, Naohiko

2018-01-01

Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification . The purified fragment was digested with trypsin to generate proteolytic peptides , and the amino acid sequences of these peptides were determined by nano-LC-mass spectrometry to identify the MT1-MMP-mediated cleavage site(s) of EphA2.

Poly(A)-tag deep sequencing data processing to extract poly(A) sites.

PubMed

Wu, Xiaohui; Ji, Guoli; Li, Qingshun Quinn

2015-01-01

Polyadenylation [poly(A)] is an essential posttranscriptional processing step in the maturation of eukaryotic mRNA. The advent of next-generation sequencing (NGS) technology has offered feasible means to generate large-scale data and new opportunities for intensive study of polyadenylation, particularly deep sequencing of the transcriptome targeting the junction of 3'-UTR and the poly(A) tail of the transcript. To take advantage of this unprecedented amount of data, we present an automated workflow to identify polyadenylation sites by integrating NGS data cleaning, processing, mapping, normalizing, and clustering. In this pipeline, a series of Perl scripts are seamlessly integrated to iteratively map the single- or paired-end sequences to the reference genome. After mapping, the poly(A) tags (PATs) at the same genome coordinate are grouped into one cleavage site, and the internal priming artifacts removed. Then the ambiguous region is introduced to parse the genome annotation for cleavage site clustering. Finally, cleavage sites within a close range of 24 nucleotides and from different samples can be clustered into poly(A) clusters. This procedure could be used to identify thousands of reliable poly(A) clusters from millions of NGS sequences in different tissues or treatments.

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

PubMed Central

Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

2014-01-01

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. PMID:24448449

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

PubMed Central

Kwong, M. Y.; Harris, R. J.

1994-01-01

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases.

PubMed

Zhu, Lihua Julie; Lawrence, Michael; Gupta, Ankit; Pagès, Hervé; Kucukural, Alper; Garber, Manuel; Wolfe, Scot A

2017-05-15

Genome editing technologies developed around the CRISPR-Cas9 nuclease system have facilitated the investigation of a broad range of biological questions. These nucleases also hold tremendous promise for treating a variety of genetic disorders. In the context of their therapeutic application, it is important to identify the spectrum of genomic sequences that are cleaved by a candidate nuclease when programmed with a particular guide RNA, as well as the cleavage efficiency of these sites. Powerful new experimental approaches, such as GUIDE-seq, facilitate the sensitive, unbiased genome-wide detection of nuclease cleavage sites within the genome. Flexible bioinformatics analysis tools for processing GUIDE-seq data are needed. Here, we describe an open source, open development software suite, GUIDEseq, for GUIDE-seq data analysis and annotation as a Bioconductor package in R. The GUIDEseq package provides a flexible platform with more than 60 adjustable parameters for the analysis of datasets associated with custom nuclease applications. These parameters allow data analysis to be tailored to different nuclease platforms with different length and complexity in their guide and PAM recognition sequences or their DNA cleavage position. They also enable users to customize sequence aggregation criteria, and vary peak calling thresholds that can influence the number of potential off-target sites recovered. GUIDEseq also annotates potential off-target sites that overlap with genes based on genome annotation information, as these may be the most important off-target sites for further characterization. In addition, GUIDEseq enables the comparison and visualization of off-target site overlap between different datasets for a rapid comparison of different nuclease configurations or experimental conditions. For each identified off-target, the GUIDEseq package outputs mapped GUIDE-Seq read count as well as cleavage score from a user specified off-target cleavage score prediction algorithm permitting the identification of genomic sequences with unexpected cleavage activity. The GUIDEseq package enables analysis of GUIDE-data from various nuclease platforms for any species with a defined genomic sequence. This software package has been used successfully to analyze several GUIDE-seq datasets. The software, source code and documentation are freely available at http://www.bioconductor.org/packages/release/bioc/html/GUIDEseq.html .

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

PubMed

Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

2000-05-01

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

Evolutionary mechanisms involved in the virulence of infectious salmon anaemia virus (ISAV), a piscine orthomyxovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Markussen, Turhan; Jonassen, Christine Monceyron; Numanovic, Sanela

2008-05-10

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing a multisystemic, emerging disease in Atlantic salmon. Here we present, for the first time, detailed sequence analyses of the full-genome sequence of a presumed avirulent isolate displaying a full-length hemagglutinin-esterase (HE) gene (HPR0), and compare this with full-genome sequences of 11 Norwegian ISAV isolates from clinically diseased fish. These analyses revealed the presence of a virulence marker right upstream of the putative cleavage site R{sub 267} in the fusion (F) protein, suggesting a Q{sub 266} {yields} L{sub 266} substitution to be a prerequisite for virulence. To gain virulence in isolates lackingmore » this substitution, a sequence insertion near the cleavage site seems to be required. This strongly suggests the involvement of a protease recognition pattern at the cleavage site of the fusion protein as a determinant of virulence, as seen in highly pathogenic influenza A virus H5 or H7 and the paramyxovirus Newcastle disease virus.« less

NMR and enzymology of modified DNA/protein interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kennedy, M.A.

1994-12-31

We have found distinct DNA structure and base dynamics precisely at the TpA cleavage site in the TTTAAA AHA III endonuclease restriction sequence. Hence, the unusual base stacking and mobility found in this sequence may be important to the mechanism of enzymatic cleavage of the phophodiester bond.

Preferential cleavage sites for Sau3A restriction endonuclease in human ribosomal DNA.

PubMed

Kupriyanova, N S; Kirilenko, P M; Netchvolodov, K K; Ryskov, A P

2000-07-21

Previous studies of cloned ribosomal DNA (rDNA) variants isolated from the cosmid library of human chromosome 13 have revealed some disproportion in representativity of different rDNA regions (N. S. Kupriyanova, K. K. Netchvolodov, P. M. Kirilenko, B. I. Kapanadze, N. K. Yankovsky, and A. P. Ryskov, Mol. Biol. 30, 51-60, 1996). Here we show nonrandom cleavage of human rDNA with Sau3A or its isoshizomer MboI under mild hydrolysis conditions. The hypersensitive cleavage sites were found to be located in the ribosomal intergenic spacer (rIGS), especially in the regions of about 5-5.5 and 11 kb upstream of the rRNA transcription start point. This finding is based on sequencing mapping of the rDNA insert ends in randomly selected cosmid clones of human chromosome 13 and on the data of digestion kinetics of cloned and noncloned human genomic rDNA with Sau3A and MboI. The results show that a methylation status and superhelicity state of the rIGS have no effect on cleavage site sensitivity. It is interesting that all primary cleavage sites are adjacent to or entering into Alu or Psi cdc 27 retroposons of the rIGS suggesting a possible role of neighboring sequences in nuclease accessibility. The results explain nonequal representation of rDNA sequences in the human genomic DNA library used for this study. Copyright 2000 Academic Press.

Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme.

PubMed Central

Ohmichi, T; Okumoto, Y; Sugimoto, N

1998-01-01

Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGUCC, binds to an RNA substrate, GGACC downward arrowGAGCCAG, cleaving the RNA substrate at one site. We have investigated the effect of the substrate sequence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadzyme acted as a catalyst for single site cleavage of a C5 deletion mutant substrate, GGAC downward arrowGAGCCAG, as well as the wild-type substrate. However, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved. Kinetic studies by surface plasmon resonance indicated that the difference between active and inactive structures reflected the slow association and dissociation rate constants of complex formation induced by Pb2+rather than differences in complex stability. CD spectra showed that the active form of the substrate-leadzyme complex was rearranged by Pb2+binding. The G8 of the wild-type substrate, which was absent in the inactive complex, is not near the cleavage site. Thus, these results show that the active substrate-leadzyme complex has a Pb2+binding site at the junction between the unpaired region (asymmetric internal loop) and the stem region, which is distal to the cleavage site. Pb2+may play a role in rearranging the bases in the asymmetric internal loop to the correct position for catalysis. PMID:9837996

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

PubMed

Kristensen, Thea; Normann, Preben; Gullberg, Maria; Fahnøe, Ulrik; Polacek, Charlotta; Rasmussen, Thomas Bruun; Belsham, Graham J

2017-03-01

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Detection of nucleic acids by multiple sequential invasive cleavages

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

1999-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Detection of nucleic acids by multiple sequential invasive cleavages 02

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

2002-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Detection of nucleic acids by multiple sequential invasive cleavages

DOEpatents

Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

2012-10-16

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Another heritage from the RNA world: self-excision of intron sequence from nuclear pre-tRNAs.

PubMed

Weber, U; Beier, H; Gross, H J

1996-06-15

The intervening sequences of nuclear tRNA precursors are known to be excised by tRNA splicing endonuclease. We show here that a T7 transcript corresponding to a pre-tRNA(Tyr) from Arabidopsis thaliana has a highly specific activity for autolytic intron excision. Self-cleavage occurs precisely at the authentic 3'-splice site and at the phosphodiester bond one nucleotide downstream of the authentic 5'-splice site. The reaction results in fragments with 2',3'-cyclic phosphate and 5'-OH termini. It is resistant to proteinase K and/or SDS treatment and is not inhibited by added tRNA. The self-cleavage depends on Mg2+ and is stimulated by spermine and Triton X-100. A set of sequence variants at the cleavage sites has been analysed for autolytic intron excision and, in parallel, for enzymatic in vitro splicing in wheat germ S23 extract. Single-stranded loops are a prerequisite for both reactions. Self-cleavage not only occurs at pyrimidine-A but also at U-U bonds. Since intron self-excision is only about five times slower than the enzymatic intron excision in a wheat germ S23 extract, we propose that the splicing endonuclease may function by improving the preciseness and efficiency of an inherent pre-tRNA self-cleavage activity.

Flanking signal and mature peptide residues influence signal peptide cleavage

PubMed Central

Choo, Khar Heng; Ranganathan, Shoba

2008-01-01

Background Signal peptides (SPs) mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I), and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i) eukaryotes (Euk) (ii) Gram-positive (Gram+) bacteria, and (iii) Gram-negative (Gram-) bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs. PMID:19091014

The proteolytic processing site of the precursor of lysyl oxidase.

PubMed Central

Cronshaw, A D; Fothergill-Gilmore, L A; Hulmes, D J

1995-01-01

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region. PMID:7864821

Characterization of a native hammerhead ribozyme derived from schistosomes

PubMed Central

OSBORNE, EDITH M.; SCHAAK, JANELL E.; DEROSE, VICTORIA J.

2005-01-01

A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop–loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop–loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop–loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of Rp-phosphorothioate substitutions at the cleavage site and 5′ to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5′ to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence. PMID:15659358

RNase L targets distinct sites in influenza A virus RNAs.

PubMed

Cooper, Daphne A; Banerjee, Shuvojit; Chakrabarti, Arindam; García-Sastre, Adolfo; Hesselberth, Jay R; Silverman, Robert H; Barton, David J

2015-03-01

Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Effect of fusion protein cleavage site sequence on generation of a genotype VII Newcastle disease virus vaccine.

PubMed

Manoharan, Vinoth K; Varghese, Berin P; Paldurai, Anandan; Samal, Siba K

2018-01-01

Newcastle disease (ND) causes severe economic loss to poultry industry worldwide. Frequent outbreaks of ND in commercial chickens vaccinated with live vaccines suggest a need to develop improved vaccines that are genetically matched against circulating Newcastle disease virus (NDV) strains. In this study, the fusion protein cleavage site (FPCS) sequence of NDV strain Banjarmasin/010 (Banj), a genotype VII NDV, was individually modified using primer mutagenesis to those of avian paramyxovirus (APMV) serotypes 2, 7 and 8 and compared with the recombinant Banjarmasin (rBanj) with avirulent NDV LaSota cleavage site (rBanj-LaSota). These FPCS mutations changed the in vitro cell-to-cell fusion activity and made rBanj FPCS mutant viruses highly attenuated in chickens. When chickens immunized with the rBanj FPCS mutant viruses and challenged with the virulent Banj, there was reduced challenge virus shedding observed compared to chickens immunized with the heterologous vaccine strain LaSota. Among the genotype VII NDV Banj vaccine candidates, rBanj-LaSota and rBanj containing FPCS of APMV-8 induced highest neutralizing antibody titers and protected chickens with reduced challenge virus shedding. These results show the effect of the F protein cleavage site sequence in generating genotype VII matched NDV vaccines.

A single amino acid change, Q114R, in the cleavage-site sequence of Newcastle disease virus fusion protein attenuates viral replication and pathogenicity.

PubMed

Samal, Sweety; Kumar, Sachin; Khattar, Sunil K; Samal, Siba K

2011-10-01

A key determinant of Newcastle disease virus (NDV) virulence is the amino acid sequence at the fusion (F) protein cleavage site. The NDV F protein is synthesized as an inactive precursor, F(0), and is activated by proteolytic cleavage between amino acids 116 and 117 to produce two disulfide-linked subunits, F(1) and F(2). The consensus sequence of the F protein cleavage site of virulent [(112)(R/K)-R-Q-(R/K)-R↓F-I(118)] and avirulent [(112)(G/E)-(K/R)-Q-(G/E)-R↓L-I(118)] strains contains a conserved glutamine residue at position 114. Recently, some NDV strains from Africa and Madagascar were isolated from healthy birds and have been reported to contain five basic residues (R-R-R-K-R↓F-I/V or R-R-R-R-R↓F-I/V) at the F protein cleavage site. In this study, we have evaluated the role of this conserved glutamine residue in the replication and pathogenicity of NDV by using the moderately pathogenic Beaudette C strain and by making Q114R, K115R and I118V mutants of the F protein in this strain. Our results showed that changing the glutamine to a basic arginine residue reduced viral replication and attenuated the pathogenicity of the virus in chickens. The pathogenicity was further reduced when the isoleucine at position 118 was substituted for valine.

Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI.

PubMed

Greene, P J; Ballard, B T; Stephenson, F; Kohr, W J; Rodriguez, H; Rosenberg, J M; Boyer, H W

1988-08-15

Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.

The highly pathogenic H7N3 avian influenza strain from July 2012 in Mexico acquired an extended cleavage site through recombination with host 28S rRNA.

PubMed

Maurer-Stroh, Sebastian; Lee, Raphael T C; Gunalan, Vithiagaran; Eisenhaber, Frank

2013-05-01

A characteristic difference between highly and non-highly pathogenic avian influenza strains is the presence of an extended, often multibasic, cleavage motif insertion in the hemagglutinin protein. Such motif is found in H7N3 strains from chicken farm outbreaks in 2012 in Mexico. Through phylogenetic, sequence and structural analysis, we try to shed light on the role, prevalence, likelihood of appearance and origin of the inserted cleavage motifs in these H7N3 avian influenza strains. The H7N3 avian influenza strain which caused outbreaks in chicken farms in June/July 2012 in Mexico has a new extended cleavage site which is the likely reason for its high pathogenicity in these birds. This cleavage site appears to have been naturally acquired and was not present in the closest low pathogenic precursors. Structural modeling shows that insertion of a productive cleavage site is quite flexible to accept insertions of different length and with sequences from different possible origins. Different from recent cleavage site insertions, the origin of the insert here is not from the viral genome but from host 28S ribosomal RNA (rRNA) instead. This is a novelty for a natural acquisition as a similar insertion has so far only been observed in a laboratory strain before. Given the abundance of viral and host RNA in infected cells, the acquisition of a pathogenicity-enhancing extended cleavage site through a similar route by other low-pathogenic avian strains in future does not seem unlikely. Important for surveillance of these H7N3 strains, the structural sites known to enhance mammalian airborne transmission are dominated by the characteristic avian residues and the risk of human to human transmission should currently be low but should be monitored for future changes accordingly. This highly pathogenic H7N3 avian influenza strain acquired a novel extended cleavage site which likely originated from recombination with 28S rRNA from the avian host. Notably, this new virus can infect humans but currently lacks critical host receptor adaptations that would facilitate human to human transmission.

Cleavage sites within the poliovirus capsid protein precursors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larsen, G.R.; Anderson, C.W.; Dorner, A.J.

1982-01-01

Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occurmore » between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.« less

Knowledge-transfer learning for prediction of matrix metalloprotease substrate-cleavage sites.

PubMed

Wang, Yanan; Song, Jiangning; Marquez-Lago, Tatiana T; Leier, André; Li, Chen; Lithgow, Trevor; Webb, Geoffrey I; Shen, Hong-Bin

2017-07-18

Matrix Metalloproteases (MMPs) are an important family of proteases that play crucial roles in key cellular and disease processes. Therefore, MMPs constitute important targets for drug design, development and delivery. Advanced proteomic technologies have identified type-specific target substrates; however, the complete repertoire of MMP substrates remains uncharacterized. Indeed, computational prediction of substrate-cleavage sites associated with MMPs is a challenging problem. This holds especially true when considering MMPs with few experimentally verified cleavage sites, such as for MMP-2, -3, -7, and -8. To fill this gap, we propose a new knowledge-transfer computational framework which effectively utilizes the hidden shared knowledge from some MMP types to enhance predictions of other, distinct target substrate-cleavage sites. Our computational framework uses support vector machines combined with transfer machine learning and feature selection. To demonstrate the value of the model, we extracted a variety of substrate sequence-derived features and compared the performance of our method using both 5-fold cross-validation and independent tests. The results show that our transfer-learning-based method provides a robust performance, which is at least comparable to traditional feature-selection methods for prediction of MMP-2, -3, -7, -8, -9 and -12 substrate-cleavage sites on independent tests. The results also demonstrate that our proposed computational framework provides a useful alternative for the characterization of sequence-level determinants of MMP-substrate specificity.

Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

PubMed

Freimuth, P; Anderson, C W

1993-03-01

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)*

PubMed Central

van der Post, Sjoerd; Subramani, Durai B.; Bäckström, Malin; Johansson, Malin E. V.; Vester-Christensen, Malene B.; Mandel, Ulla; Bennett, Eric P.; Clausen, Henrik; Dahlén, Gunnar; Sroka, Aneta; Potempa, Jan; Hansson, Gunnar C.

2013-01-01

The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation. PMID:23546879

Nucleic acid detection kits

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann; Kwiatkowski, Robert W.; Vavra, Stephanie H.

2005-03-29

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA.

PubMed

Razvi, F; Gargiulo, G; Worcel, A

1983-08-01

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

PubMed

Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

2016-11-01

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-02-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed Central

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-01-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Semler, B.L.; Anderson, C.W.; Kitamura, N.

1981-06-01

A partial amino-terminal amino acid sequence of each of the major proteins encoded by the replicase region of the poliovirus genome has been determined. A comparison of this sequence information with the amino acid sequence predicted from the RNA sequence that has been determined for the 3' region of the poliovirus genome has allowed us to locate precisely the proteolytic cleavage sites at which the initial polyprotein is processed to create the poliovirus products P3-1b (NCVP1b), P3-2 (NCVP2), P3-4b (NCVP4b), and P3-7c (NCVP7c). For each of these products, as well as for the small genome-linked protein VPg, proteolytic cleavage occursmore » between a glutamine and a glycine residue to create the amino terminus of each protein. This result suggests that a single proteinase may be responsible for all of these cleavages. The sequence data also allow the precise positioning of the genome-linked protein VPg within the precursor P3-1b just proximal to the amino terminus of polypeptide P3-2.« less

The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

PubMed

Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

2016-11-15

Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme. Copyright © 2016 Elsevier Ltd. All rights reserved.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

PubMed

O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

Programmable RNA recognition and cleavage by CRISPR/Cas9

PubMed Central

O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

2014-01-01

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition. PMID:25274302

Characterization of clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses from wild birds possessing atypical hemagglutinin polybasic cleavage sites.

PubMed

Usui, Tatsufumi; Soda, Kosuke; Tomioka, Yukiko; Ito, Hiroshi; Yabuta, Toshiyo; Takakuwa, Hiroki; Otsuki, Koichi; Ito, Toshihiro; Yamaguchi, Tsuyoshi

2017-02-01

Since 2014, clade 2.3.4.4 H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have been distributed worldwide. These viruses, which were reported to be highly virulent in chickens by intravenous inoculation, have a consensus HPAI motif PLRERRRKR at the HA cleavage site. However, two-clade 2.3.4.4 H5N8 viruses which we isolated from wild migratory birds in late 2014 in Japan possessed atypical HA cleavage sequences. A swan isolate, Tottori/C6, had a novel polybasic cleavage sequence, PLGERRRKR, and another isolate from a dead mandarin duck, Gifu/01, had a heterogeneous mixture of consensus PLRERRRKR and variant PLRERRRRKR sequences. The polybasic HA cleavage site is the prime virulence determinant of AIVs. Therefore, in the present study, we examined the pathogenicity of these H5N8 isolates in chickens by intravenous inoculation. When 10 6 EID 50 of these viruses were intravenously inoculated into chickens, the mean death time associated with Tottori/C6 was substantially longer (>6.1 days) than that associated with Gifu/01 (2.5 days). These viruses had comparable abilities to replicate in tissue culture cells in the presence and absence of exogenous trypsin, but the growth of Tottori/C6 was hampered. These results indicate that the novel cleavage motif of Tottori/C6 did not directly affect the infectivity of the virus, but Tottori/C6 caused attenuated pathogenicity in chickens because of hampered replication efficiency. It is important to test for the emergence of diversified HPAIVs, because introduction of HPAIVs with a lower virulence like Tottori/C6 might hinder early detection of affected birds in poultry farms.

Variable context Markov chains for HIV protease cleavage site prediction.

PubMed

Oğul, Hasan

2009-06-01

Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites

PubMed Central

Gratias, Ariane; Lepère, Gersende; Garnier, Olivier; Rosa, Sarah; Duharcourt, Sandra; Malinsky, Sophie; Meyer, Eric; Bétermier, Mireille

2008-01-01

Somatic genome assembly in the ciliate Paramecium involves the precise excision of thousands of short internal eliminated sequences (IESs) that are scattered throughout the germline genome and often interrupt open reading frames. Excision is initiated by double-strand breaks centered on the TA dinucleotides that are conserved at each IES boundary, but the factors that drive cleavage site recognition remain unknown. A degenerate consensus was identified previously at IES ends and genetic analyses confirmed the participation of their nucleotide sequence in efficient excision. Even for wild-type IESs, however, variant excision patterns (excised or nonexcised) may be inherited maternally through sexual events, in a homology-dependent manner. We show here that this maternal epigenetic control interferes with the targeting of DNA breaks at IES ends. Furthermore, we demonstrate that a mutation in the TA at one end of an IES impairs DNA cleavage not only at the mutant end but also at the wild-type end. We conclude that crosstalk between both ends takes place prior to their cleavage and propose that the ability of an IES to adopt an excision-prone conformation depends on the combination of its nucleotide sequence and of additional determinants. PMID:18420657

Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

2014-04-15

In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies.more » The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.« less

Nucleotide sequence of the 3' terminal region of lettuce mosaic potyvirus RNA shows a Gln/Val dipeptide at the cleavage site between the polymerase and the coat protein.

PubMed

Dinant, S; Lot, H; Albouy, J; Kuziak, C; Meyer, M; Astier-Manifacier, S

1991-01-01

DNA complementary to the 3' terminal 1651 nucleotides of the genome of the common strain of lettuce mosaic virus (LMV-O) has been cloned and sequenced. Microsequencing of the N-terminus enabled localization of the coat protein gene in this sequence. It showed also that the LMV coat protein coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein by cleavage at an unusual Q/V dipeptide between the polymerase and the coat protein. This is the first report of such a site for cleavage of a potyvirus polyprotein, where only Q/A, Q/S, and Q/G cleavage sites have been reported. The LMV coat protein gene encodes a 278 amino acid polypeptide with a calculated Mr of 31,171 and is flanked by a region which has a high degree of homology with the putative polymerase and a 3' untranslated region of 211 nucleotides in length. Percentage of homology with the coat protein of other potyviruses confirms that LMV is a distinct member of this group. Moreover, amino acid homologies noticed with the coat protein of potexvirus, bymovirus, and carlavirus elongated plant viruses suggest a functional significance for the conserved domains.

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses.

PubMed

Davis, David A; Naiman, Nicole E; Wang, Victoria; Shrestha, Prabha; Haque, Muzammel; Hu, Duosha; Anagho, Holda A; Carey, Robert F; Davidoff, Katharine S; Yarchoan, Robert

2015-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1 beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.

In silico Analysis of 3′-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data

PubMed Central

Tanaka, Mizuki; Sakai, Yoshifumi; Yamada, Osamu; Shintani, Takahiro; Gomi, Katsuya

2011-01-01

To investigate 3′-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3′-untranslated region (3′ UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3′ UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3′ UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15–30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3′-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3′-end-processing signals are similar to those in yeast and plants, some notable differences exist between them. PMID:21586533

alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases.

PubMed Central

Miller, P B; Wakarchuk, W W; Warren, R A

1985-01-01

The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase. PMID:2987859

Substrate recognition by ribonucleoprotein ribonuclease MRP

PubMed Central

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S.

2011-01-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5′ ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed. PMID:21173200

Substrate recognition by ribonucleoprotein ribonuclease MRP.

PubMed

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S

2011-02-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein.

PubMed

Li, Yifeng

2012-02-01

LL-37 is the only cathelicidin-derived antimicrobial peptide found in humans and it has a multifunctional role in host defense. The peptide has been shown to possess immunomodulatory functions in addition to antimicrobial activity. To provide sufficient material for biological and structural characterization of this important peptide, various systems were developed to produce recombinant LL-37 in Escherichia coli. In one previous approach, LL-37 coding sequence was cloned into vector pET-32a, allowing the peptide to be expressed as a thioredoxin fusion. The fusion protein contains two thrombin cleavage sites: a vector-encoded one that is 30-residue upstream of the insert and an engineered one that is immediately adjacent to LL-37. Cleavage at these two sites shall generate three fragments, one of which is the target peptide. However, when the fusion protein was treated with thrombin, cleavage only occurred at the remote upstream site. A plausible explanation is that the thrombin site adjacent to LL-37 is less accessible due to the peptide's aggregation tendency and cleavage at the remote site generates a fragment, which forms a large aggregate that buries the intended site. In this study, I deleted the vector-encoded thrombin site and S tag in pET-32a, and then inserted the coding sequence for LL-37 plus a thrombin site into the modified vector. Although removing the S tag did not change the oligomeric state of the fusion protein, deletion of the vector-encoded thrombin site allowed the fusion to be cleaved at the engineered site to release LL-37. The released peptide was separated from the carrier and cleavage enzyme by size-exclusion chromatography. This new approach enables a quick production of high quality active LL-37 with a decent amount. Copyright © 2011 Elsevier Inc. All rights reserved.

SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula

PubMed Central

Wong, Emily S. W.; Hardy, Margaret C.; Wood, David; Bailey, Timothy; King, Glenn F.

2013-01-01

Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree) and developed an algorithm (SpiderP) for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM) framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor) from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP) is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html), a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from the SpiderP website. PMID:23894279

msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding

PubMed Central

Gilad, Yoav; Pritchard, Jonathan K.; Stephens, Matthew

2015-01-01

Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede. PMID:26406244

msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

PubMed

Raj, Anil; Shim, Heejung; Gilad, Yoav; Pritchard, Jonathan K; Stephens, Matthew

2015-01-01

Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

PubMed Central

Xu, J; Mendez, E; Caron, P R; Lin, C; Murcko, M A; Collett, M S; Rice, C M

1997-01-01

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication. PMID:9188600

Effect of pyrimido[1,6-a]benzimidazoles, quinolones, and Ca2+ on the DNA gyrase-mediated cleavage reaction.

PubMed Central

Gmünder, H; Kuratli, K; Keck, W

1995-01-01

The quinolones inhibit the A subunit of DNA gyrase in the presence of Mg2+ by interrupting the DNA breakage and resealing steps, and the latter step is also retarded without quinolones if Mg2+ is replaced by Ca2+. Pyrimido[1,6-a]benzimidazoles have been found to represent a new class of potent DNA gyrase inhibitors which also act at the A subunit. To determine alterations in the DNA sequence specificity of DNA gyrase for cleavage sites in the presence of inhibitors of both classes or in the presence of Ca2+, we used DNA restriction fragments of 164, 85, and 71 bp from the pBR322 plasmid as model substrates. Each contained, at a different position, the 20-bp pBR322 sequence around position 990, where DNA gyrase preferentially cleaves in the presence of quinolones. Our results show that pyrimido[1,6-a]benzimidazoles have a mode of action similar to that of quinolones; they inhibit the resealing step and influence the DNA sequence specificity of DNA gyrase in the same way. Differences between inhibitors of both classes could be observed only in the preferences of DNA gyrase for these cleavage sites. The 20-bp sequence appeared to have some properties that induced DNA gyrase to cleave all three DNA fragments in the presence of inhibitors within this sequence, whereas cleavage in the presence of Ca2+ was in addition dependent on the length of the DNA fragments. PMID:7695300

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

PubMed

Kolev, Nikolay G; Hartland, Emilia I; Huber, Paul W

2008-10-01

The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

PubMed

Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

2006-11-21

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

PolyA_DB 3 catalogs cleavage and polyadenylation sites identified by deep sequencing in multiple genomes

PubMed Central

Wang, Ruijia; Nambiar, Ram; Zheng, Dinghai

2018-01-01

Abstract PolyA_DB is a database cataloging cleavage and polyadenylation sites (PASs) in several genomes. Previous versions were based mainly on expressed sequence tags (ESTs), which had a limited amount and could lead to inaccurate PAS identification due to the presence of internal A-rich sequences in transcripts. Here, we present an updated version of the database based solely on deep sequencing data. First, PASs are mapped by the 3′ region extraction and deep sequencing (3′READS) method, ensuring unequivocal PAS identification. Second, a large volume of data based on diverse biological samples increases PAS coverage by 3.5-fold over the EST-based version and provides PAS usage information. Third, strand-specific RNA-seq data are used to extend annotated 3′ ends of genes to obtain more thorough annotations of alternative polyadenylation (APA) sites. Fourth, conservation information of PAS across mammals sheds light on significance of APA sites. The database (URL: http://www.polya-db.org/v3) currently holds PASs in human, mouse, rat and chicken, and has links to the UCSC genome browser for further visualization and for integration with other genomic data. PMID:29069441

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

PubMed Central

Jablonski, Joseph; Clementz, Mark; Ryan, Kevin; Valente, Susana T.

2014-01-01

The 3’ end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3’ end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5’ cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3’ end processing of HIV-1 mRNAs. PMID:24835792

LBSizeCleav: improved support vector machine (SVM)-based prediction of Dicer cleavage sites using loop/bulge length.

PubMed

Bao, Yu; Hayashida, Morihiro; Akutsu, Tatsuya

2016-11-25

Dicer is necessary for the process of mature microRNA (miRNA) formation because the Dicer enzyme cleaves pre-miRNA correctly to generate miRNA with correct seed regions. Nonetheless, the mechanism underlying the selection of a Dicer cleavage site is still not fully understood. To date, several studies have been conducted to solve this problem, for example, a recent discovery indicates that the loop/bulge structure plays a central role in the selection of Dicer cleavage sites. In accordance with this breakthrough, a support vector machine (SVM)-based method called PHDCleav was developed to predict Dicer cleavage sites which outperforms other methods based on random forest and naive Bayes. PHDCleav, however, tests only whether a position in the shift window belongs to a loop/bulge structure. In this paper, we used the length of loop/bulge structures (in addition to their presence or absence) to develop an improved method, LBSizeCleav, for predicting Dicer cleavage sites. To evaluate our method, we used 810 empirically validated sequences of human pre-miRNAs and performed fivefold cross-validation. In both 5p and 3p arms of pre-miRNAs, LBSizeCleav showed greater prediction accuracy than PHDCleav did. This result suggests that the length of loop/bulge structures is useful for prediction of Dicer cleavage sites. We developed a novel algorithm for feature space mapping based on the length of a loop/bulge for predicting Dicer cleavage sites. The better performance of our method indicates the usefulness of the length of loop/bulge structures for such predictions.

Development of a Novel Anti-HIV-1 Agent from within: Effect of Chimeric Vpr-Containing Protease Cleavage Site Residues on Virus Replication

NASA Astrophysics Data System (ADS)

Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

1997-04-01

Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

Identification of an Electrostatic Ruler Motif for Sequence-Specific Binding of Collagenase to Collagen.

PubMed

Subramanian, Sundar Raman; Singam, Ettayapuram Ramaprasad Azhagiya; Berinski, Michael; Subramanian, Venkatesan; Wade, Rebecca C

2016-08-25

Sequence-specific cleavage of collagen by mammalian collagenase plays a pivotal role in cell function. Collagenases are matrix metalloproteinases that cleave the peptide bond at a specific position on fibrillar collagen. The collagenase Hemopexin-like (HPX) domain has been proposed to be responsible for substrate recognition, but the mechanism by which collagenases identify the cleavage site on fibrillar collagen is not clearly understood. In this study, Brownian dynamics simulations coupled with atomic-detail and coarse-grained molecular dynamics simulations were performed to dock matrix metalloproteinase-1 (MMP-1) on a collagen IIIα1 triple helical peptide. We find that the HPX domain recognizes the collagen triple helix at a conserved R-X11-R motif C-terminal to the cleavage site to which the HPX domain of collagen is guided electrostatically. The binding of the HPX domain between the two arginine residues is energetically stabilized by hydrophobic contacts with collagen. From the simulations and analysis of the sequences and structural flexibility of collagen and collagenase, a mechanistic scheme by which MMP-1 can recognize and bind collagen for proteolysis is proposed.

Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach

PubMed Central

Ai, X; Butts, B; Vora, K; Li, W; Tache-Talmadge, C; Fridman, A; Mehmet, H

2011-01-01

Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity. PMID:21881607

Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

PubMed Central

Jayasena, S D; Johnston, B H

1992-01-01

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo. Images PMID:1565648

Target mimicry provides a new mechanism for regulation of microRNA activity.

PubMed

Franco-Zorrilla, José Manuel; Valli, Adrián; Todesco, Marco; Mateos, Isabel; Puga, María Isabel; Rubio-Somoza, Ignacio; Leyva, Antonio; Weigel, Detlef; García, Juan Antonio; Paz-Ares, Javier

2007-08-01

MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site. Here, we examined the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION 1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (Pi) starvation-induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term 'target mimicry' to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics.

Evaluation of the Contributions of Individual Viral Genes to Newcastle Disease Virus Virulence and Pathogenesis

PubMed Central

Paldurai, Anandan; Kim, Shin-Hee; Nayak, Baibaswata; Xiao, Sa; Shive, Heather; Collins, Peter L.

2014-01-01

ABSTRACT Naturally occurring Newcastle disease virus (NDV) strains vary greatly in virulence. The presence of multibasic residues at the proteolytic cleavage site of the fusion (F) protein has been shown to be a primary determinant differentiating virulent versus avirulent strains. However, there is wide variation in virulence among virulent strains. There also are examples of incongruity between cleavage site sequence and virulence. These observations suggest that additional viral factors contribute to virulence. In this study, we evaluated the contribution of each viral gene to virulence individually and in different combinations by exchanging genes between velogenic (highly virulent) strain GB Texas (GBT) and mesogenic (moderately virulent) strain Beaudette C (BC). These two strains are phylogenetically closely related, and their F proteins contain identical cleavage site sequences, 112RRQKR↓F117. A total of 20 chimeric viruses were constructed and evaluated in vitro, in 1-day-old chicks, and in 2-week-old chickens. The results showed that both the envelope-associated and polymerase-associated proteins contribute to the difference in virulence between rBC and rGBT, with the envelope-associated proteins playing the greater role. The F protein was the major individual contributor and was sometimes augmented by the homologous M and HN proteins. The dramatic effect of F was independent of its cleavage site sequence since that was identical in the two strains. The polymerase L protein was the next major individual contributor and was sometimes augmented by the homologous N and P proteins. The leader and trailer regions did not appear to contribute to the difference in virulence between BC and GBT. IMPORTANCE This study is the first comprehensive and systematic study of NDV virulence and pathogenesis. Genetic exchanges between a mesogenic and a velogenic strain revealed that the fusion glycoprotein is the major virulence determinant regardless of the identical virulence protease cleavage site sequence present in both strains. The contribution of the large polymerase protein to NDV virulence is second only to that of the fusion glycoprotein. The identification of virulence determinants is of considerable importance, because of the potential to generate better live attenuated NDV vaccines. It may also be possible to apply these findings to other paramyxoviruses. PMID:24850737

SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing.

PubMed

Yu, Simei; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Jain, Shruti; Rolicka, Anna; Östlund Farrants, Ann-Kristin; Visa, Neus

2018-05-31

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA

DOE PAGES

Gordon, Gina C.; Cameron, Jeffrey C.; Pfleger, Brian F.

2017-03-28

Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage of Escherichia coli and a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found in aceEF, proP, tnaC, dctA, pheM, sdhC, yhhQ, glpT, aceK,more » and gluQ accelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in the ahpF, pflB, and yajQ transcripts led to stabilized secondary transcripts. Two other novel sites in hisL and pheM overlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in an rnc deletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism. IMPORTANCE: The concerted action and overlapping functions of endoribonucleases, exoribonucleases, and RNA processing enzymes complicate the study of global RNA turnover and recycling of specific transcripts. More information about RNase specificity and activity is needed to make predictions of transcript half-life and to design synthetic transcripts with optimal stability. RNase III does not have a conserved target sequence but instead recognizes RNA secondary structure. Prior to this study, only a few RNase III target sites in E. coli were known, so we used RNA sequencing to provide a more comprehensive list of cleavage sites and to examine the impact of RNase III on transcript degradation. Finally, with this added information on how RNase III participates in transcript regulation and recycling, a more complete picture of RNA turnover can be developed for E. coli. Similar approaches could be used to augment our understanding of RNA turnover in other bacteria.« less

RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gordon, Gina C.; Cameron, Jeffrey C.; Pfleger, Brian F.

Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage of Escherichia coli and a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found in aceEF, proP, tnaC, dctA, pheM, sdhC, yhhQ, glpT, aceK,more » and gluQ accelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in the ahpF, pflB, and yajQ transcripts led to stabilized secondary transcripts. Two other novel sites in hisL and pheM overlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in an rnc deletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism. IMPORTANCE: The concerted action and overlapping functions of endoribonucleases, exoribonucleases, and RNA processing enzymes complicate the study of global RNA turnover and recycling of specific transcripts. More information about RNase specificity and activity is needed to make predictions of transcript half-life and to design synthetic transcripts with optimal stability. RNase III does not have a conserved target sequence but instead recognizes RNA secondary structure. Prior to this study, only a few RNase III target sites in E. coli were known, so we used RNA sequencing to provide a more comprehensive list of cleavage sites and to examine the impact of RNase III on transcript degradation. Finally, with this added information on how RNase III participates in transcript regulation and recycling, a more complete picture of RNA turnover can be developed for E. coli. Similar approaches could be used to augment our understanding of RNA turnover in other bacteria.« less

AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination

PubMed Central

White, Eleanor; Kamieniarz-Gdula, Kinga; Dye, Michael J.; Proudfoot, Nick J.

2013-01-01

RNA Polymerase II (Pol II) termination is dependent on RNA processing signals as well as specific terminator elements located downstream of the poly(A) site. One of the two major terminator classes described so far is the Co-Transcriptional Cleavage (CoTC) element. We show that homopolymer A/T tracts within the human β-globin CoTC-mediated terminator element play a critical role in Pol II termination. These short A/T tracts, dispersed within seemingly random sequences, are strong terminator elements, and bioinformatics analysis confirms the presence of such sequences in 70% of the putative terminator regions (PTRs) genome-wide. PMID:23258704

Nucleic acid indexing

DOEpatents

Guilfoyle, Richard A.; Guo, Zhen

2001-01-01

A restriction site indexing method for selectively amplifying any fragment generated by a Class II restriction enzyme includes adaptors specific to fragment ends containing adaptor indexing sequences complementary to fragment indexing sequences near the termini of fragments generated by Class II enzyme cleavage. A method for combinatorial indexing facilitates amplification of restriction fragments whose sequence is not known.

Nucleic acid indexing

DOEpatents

Guilfoyle, Richard A.; Guo, Zhen

1999-01-01

A restriction site indexing method for selectively amplifying any fragment generated by a Class II restriction enzyme includes adaptors specific to fragment ends containing adaptor indexing sequences complementary to fragment indexing sequences near the termini of fragments generated by Class II enzyme cleavage. A method for combinatorial indexing facilitates amplification of restriction fragments whose sequence is not known.

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

PubMed

Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

2016-12-27

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

Prediction of proprotein convertase cleavage sites.

PubMed

Duckert, Peter; Brunak, Søren; Blom, Nikolaj

2004-01-01

Many secretory proteins and peptides are synthesized as inactive precursors that in addition to signal peptide cleavage undergo post-translational processing to become biologically active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created additional focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on experimental results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network. The ProP method is made publicly available at http://www.cbs.dtu.dk/services/ProP.

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity.

PubMed

Duda, Anja; Stange, Annett; Lüftenegger, Daniel; Stanke, Nicole; Westphal, Dana; Pietschmann, Thomas; Eastman, Scott W; Linial, Maxine L; Rethwilm, Axel; Lindemann, Dirk

2004-12-01

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.

HIV-1 protease-substrate coevolution in nelfinavir resistance.

PubMed

Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

2014-07-01

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Preferential binding of daunomycin to 5'ATCG and 5'ATGC sequences revealed by footprinting titration experiments.

PubMed

Chaires, J B; Herrera, J E; Waring, M J

1990-07-03

Results from a high-resolution deoxyribonuclease I (DNase I) footprinting titration procedure are described that identify preferred daunomycin binding sites within the 160 bp tyr T DNA fragment. We have obtained single-bond resolution at 65 of the 160 potential binding sites within the tyr T fragment and have examined the effect of 0-3.0 microM total daunomycin concentration on the susceptibility of these sites toward digestion by DNase I. Four types of behavior are observed: (i) protection from DNase I cleavage; (ii) protection, but only after reaching a critical total daunomycin concentration; (iii) enhanced cleavage; (iv) no effect of added drug. Ten sites were identified as the most strongly protected on the basis of the magnitude of the reduction of their digestion product band areas in the presence of daunomycin. These were identified as the preferred daunomycin binding sites. Seven of these 10 sites are found at the end of the triplet sequences 5'ATGC and 5'ATCG, where the notation AT indicates that either A or T may occupy the position. The remaining three strongly protected sites are found at the ends of the triplet sequence 5'ATCAT. Of the preferred daunomycin binding sites we identify in this study, the sequence 5'ATCG is consistent with the specificity predicted by the theoretical studies of Chen et al. [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466] and is the very sequence to which daunomycin is observed to be bound in two recent X-ray crystallographic studies. Solution studies, theoretical studies, and crystallographic studies have thus converged to provide a consistent and coherent picture of the sequence preference of this important anticancer antibiotic.

RNA-programmed genome editing in human cells

PubMed Central

Jinek, Martin; East, Alexandra; Cheng, Aaron; Lin, Steven; Ma, Enbo; Doudna, Jennifer

2013-01-01

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells. DOI: http://dx.doi.org/10.7554/eLife.00471.001 PMID:23386978

Antibody proteolysis: a common picture emerging from plants

PubMed Central

Donini, Marcello; Lombardi, Raffaele; Lonoce, Chiara; Di Carli, Mariasole; Marusic, Carla; Morea, Veronica; Di Micco, Patrizio

2015-01-01

We have recently characterized the degradation profiles of 2 human IgG1 monoclonal antibodies, the tumor-targeting mAb H10 and the anti-HIV mAb 2G12. Both mAbs were produced in plants either as stable transgenics or using a transient expression system based on leaf agroinfiltration. The purified antibodies were separated by 1DE and protein bands were characterized by N-terminal sequencing. The proteolytic cleavage sites identified in the heavy chain (HC) of both antibodies were localized in 3 inter-domain regions, suggesting that the number of proteolytic cleavage events taking place in plants is limited. One of the cleavage sites, close to the hinge region, was common to both antibodies. PMID:26186119

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.

PubMed

Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

2016-03-01

The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.

The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells

PubMed Central

Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

2016-01-01

The clustered regularly-interspaced short palindromic repeats (CRISPR)—CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications. PMID:26782639

Variola virus topoisomerase: DNA cleavage specificity and distribution of sites in Poxvirus genomes.

PubMed

Minkah, Nana; Hwang, Young; Perry, Kay; Van Duyne, Gregory D; Hendrickson, Robert; Lefkowitz, Elliot J; Hannenhalli, Sridhar; Bushman, Frederic D

2007-08-15

Topoisomerase enzymes regulate superhelical tension in DNA resulting from transcription, replication, repair, and other molecular transactions. Poxviruses encode an unusual type IB topoisomerase that acts only at conserved DNA sequences containing the core pentanucleotide 5'-(T/C)CCTT-3'. In X-ray structures of the variola virus topoisomerase bound to DNA, protein-DNA contacts were found to extend beyond the core pentanucleotide, indicating that the full recognition site has not yet been fully defined in functional studies. Here we report quantitation of DNA cleavage rates for an optimized 13 bp site and for all possible single base substitutions (40 total sites), with the goals of understanding the molecular mechanism of recognition and mapping topoisomerase sites in poxvirus genome sequences. The data allow a precise definition of enzyme-DNA interactions and the energetic contributions of each. We then used the resulting "action matrix" to show that favorable topoisomerase sites are distributed all along the length of poxvirus DNA sequences, consistent with a requirement for local release of superhelical tension in constrained topological domains. In orthopox genomes, an additional central cluster of sites was also evident. A negative correlation of predicted topoisomerase sites was seen relative to early terminators, but no correlation was seen with early or late promoters. These data define the full variola virus topoisomerase recognition site and provide a new window on topoisomerase function in vivo.

Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system.

PubMed

Liu, Xiaoxi; Homma, Ayaka; Sayadi, Jamasb; Yang, Shu; Ohashi, Jun; Takumi, Toru

2016-01-27

The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

Sequence-specific DNA cleavage by Fe2+-mediated fenton reactions has possible biological implications.

PubMed

Henle, E S; Han, Z; Tang, N; Rai, P; Luo, Y; Linn, S

1999-01-08

Preferential cleavage sites have been determined for Fe2+/H2O2-mediated oxidations of DNA. In 50 mM H2O2, preferential cleavages occurred at the nucleoside 5' to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG)81 human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H2O2, preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe2+ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe2+ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe2+ with the three purine N7 residues. Fe2+-dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A5mCGA were consistent with this model.

The Structure and Specificity of the Type III Secretion System Effector NleC Suggest a DNA Mimicry Mechanism of Substrate Recognition

PubMed Central

2015-01-01

Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221

SfiI genomic cleavage map of Escherichia coli K-12 strain MG1655.

PubMed Central

Perkins, J D; Heath, J D; Sharma, B R; Weinstock, G M

1992-01-01

An SfiI restriction map of Escherichia coli K-12 strain MG1655 is presented. The map contains thirty-one cleavage sites separating fragments ranging in size from 407 kb to 3.7 kb. Several techniques were used in the construction of this map, including CHEF pulsed field gel electrophoresis; physical analysis of a set of twenty-six auxotrophic transposon insertions; correlation with the restriction map of Kohara and coworkers using the commercially available E. coli Gene Mapping Membranes; analysis of publicly available sequence information; and correlation of the above data with the combined genetic and physical map developed by Rudd, et al. The combination of these techniques has yielded a map in which all but one site can be localized within a range of +/- 2 kb, and over half the sites can be localized precisely by sequence data. Two sites present in the EcoSeq5 sequence database are not cleaved in MG1655 and four sites are noted to be sensitive to methylation by the dcm methylase. This map, combined with the NotI physical map of MG1655, can aid in the rapid, precise mapping of several different types of genetic alterations, including transposon mediated mutations and other insertions, inversions, deletions and duplications. Images PMID:1312707

Distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the Junin virus envelope glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

York, Joanne; Nunberg, Jack H.

2007-03-01

The arenavirus envelope glycoprotein (GP-C) retains a cleaved and stable signal peptide (SSP) as an essential subunit of the mature complex. This 58-amino-acid residue peptide serves as a signal sequence and is additionally required to enable transit of the assembled GP-C complex to the Golgi, and for pH-dependent membrane fusion activity. We have investigated the C-terminal region of the Junin virus SSP to study the role of the cellular signal peptidase (SPase) in generating SSP. Site-directed mutagenesis at the cleavage site (positions - 1 and - 3) reveals a pattern of side-chain preferences consistent with those of SPase. Although positionmore » - 2 is degenerate for SPase cleavage, this residue in the arenavirus SSP is invariably a cysteine. In the Junin virus, this cysteine is not involved in disulfide bonding. We show that replacement with alanine or serine is tolerated for SPase cleavage but prevents the mutant SSP from associating with GP-C and enabling transport to the cell surface. Conversely, an arginine mutation at position - 1 that prevents SPase cleavage is fully compatible with GP-C-mediated membrane fusion activity when the mutant SSP is provided in trans. These results point to distinct roles of SSP sequences in SPase cleavage and GP-C biogenesis. Further studies of the unique structural organization of the GP-C complex will be important in identifying novel opportunities for antiviral intervention against arenaviral hemorrhagic disease.« less

[Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

PubMed

Vtiurina, N N; Grokhovskiĭ, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

2011-01-01

The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed.

Mutations altering the cleavage specificity of a homing endonuclease

PubMed Central

Seligman, Lenny M.; Chisholm, Karen M.; Chevalier, Brett S.; Chadsey, Meggen S.; Edwards, Samuel T.; Savage, Jeremiah H.; Veillet, Adeline L.

2002-01-01

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences. PMID:12202772

Role of the protein in the DNA sequence specificity of the cleavage site stabilized by the camptothecin topoisomerase IB inhibitor: a metadynamics study

PubMed Central

Coletta, Andrea; Desideri, Alessandro

2013-01-01

Camptothecin (CPT) is a topoisomerase IB (TopIB) selective inhibitor whose derivatives are currently used in cancer therapy. TopIB cleaves DNA at any sequence, but in the presence of CPT the only stabilized protein–DNA covalent complex is the one having a thymine in position −1 with respect to the cleavage site. A metadynamics simulation of two TopIB–DNA–CPT ternary complexes differing for the presence of a thymine or a cytosine in position −1 indicates the occurrence of two different drug’s unbinding pathways. The free-energy difference between the bound state and the transition state is large when a thymine is present in position −1 and is strongly reduced in presence of a cytosine, in line with the different drug stabilization properties of the two systems. Such a difference is strictly related to the changes in the hydrogen bond network between the protein, the DNA and the drug in the two systems, indicating a direct role of the protein in determining the specificity of the cleavage site sequence stabilized by the CPT. Calculations carried out in presence of one compound of the indenoisoquinoline family (NSC314622) indicate a comparable energy difference between the bound and the transition state independently of the presence of a thymine or a cytosine in position −1, in line with the experimental results. PMID:24003027

Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

PubMed

Ryan, Daniel E; Taussig, David; Steinfeld, Israel; Phadnis, Smruti M; Lunstad, Benjamin D; Singh, Madhurima; Vuong, Xuan; Okochi, Kenji D; McCaffrey, Ryan; Olesiak, Magdalena; Roy, Subhadeep; Yung, Chong Wing; Curry, Bo; Sampson, Jeffrey R; Bruhn, Laurakay; Dellinger, Douglas J

2018-01-25

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

NASA Astrophysics Data System (ADS)

Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

Truncation of the TAR DNA-binding protein 43 is not a prerequisite for cytoplasmic relocalization, and is suppressed by caspase inhibition and by introduction of the A90V sequence variant

PubMed Central

Brandon, Nicholas J.; Moss, Stephen J.

2017-01-01

The RNA-binding and -processing protein TAR DNA-binding protein 43 (TDP-43) is heavily linked to the underlying causes and pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In these diseases, TDP-43 is mislocalized, hyperphosphorylated, ubiquitinated, aggregated and cleaved. The importance of TDP-43 cleavage in the disease pathogenesis is still poorly understood. Here we detail the use of D-sorbitol as an exogenous stressor that causes TDP-43 cleavage in HeLa cells, resulting in a 35 kDa truncated product that accumulates in the cytoplasm within one hour of treatment. We confirm that the formation of this 35 kDa cleavage product is mediated by the activation of caspases. Inhibition of caspases blocks the cleavage of TDP-43, but does not prevent the accumulation of full-length protein in the cytoplasm. Using D-sorbitol as a stressor and caspase activator, we also demonstrate that the A90V variant of TDP-43, which lies adjacent to the caspase cleavage site within the nuclear localization sequence of TDP-43, confers partial resistance against caspase-mediated generation of the 35 kDa cleavage product. PMID:28510586

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway.

PubMed

Garcia, Angelo L; Han, Shan-Kuo; Janssen, William G; Khaing, Zin Z; Ito, Timothy; Glucksman, Marc J; Benson, Deanna L; Salton, Stephen R J

2005-12-16

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.

Functional dissection of the alphavirus capsid protease: sequence requirements for activity.

PubMed

Thomas, Saijo; Rai, Jagdish; John, Lijo; Günther, Stephan; Drosten, Christian; Pützer, Brigitte M; Schaefer, Stephan

2010-11-18

The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

Albumin Redhill (-1 Arg, 320 Ala yields Thr): A glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brennan, S.O.; Myles, T.; Peach, R.J.

1990-01-01

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind {sup 63}Ni{sup 2+} and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni{sup 2+} was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala {yields} Thr. Thismore » introduces as Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg {yields} Cys. The authors propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.« less

Transcriptome Wide Annotation of Eukaryotic RNase III Reactivity and Degradation Signals

PubMed Central

Gagnon, Jules; Lavoie, Mathieu; Catala, Mathieu; Malenfant, Francis; Elela, Sherif Abou

2015-01-01

Detection and validation of the RNA degradation signals controlling transcriptome stability are essential steps for understanding how cells regulate gene expression. Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome, and encompass a wide variety of sequences, indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes, and their impact on RNA expression is linked to growth conditions. Together, the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions. PMID:25680180

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-11-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed Central

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-01-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM. Images PMID:1656067

Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.

PubMed

Rousseau, Beth A; Hou, Zhonggang; Gramelspacher, Max J; Zhang, Yan

2018-03-01

The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA. Copyright © 2018 Elsevier Inc. All rights reserved.

Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

DOE PAGES

Horton, J. R.; Wang, H.; Mabuchi, M. Y.; ...

2014-09-27

MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNAmore » molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.« less

A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease.

PubMed

Oppliger, Joel; da Palma, Joel Ramos; Burri, Dominique J; Bergeron, Eric; Khatib, Abdel-Majid; Spiropoulou, Christina F; Pasquato, Antonella; Kunz, Stefan

2016-01-15

Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus infection of human cells is the processing of the viral envelope glycoprotein by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). In order to break the species barrier during zoonotic transmission and cause severe disease in humans, newly emerging arenaviruses must be able to hijack human SKI-1/S1P efficiently. Here we implement a newly developed cell-based molecular sensor for human SKI-1/S1P to characterize the processing of arenavirus glycoproteins in a quantitative manner. We further use our sensor to correctly predict efficient processing of the glycoprotein of the newly emergent pathogenic Lujo virus by human SKI-1/S1P. Our sensor thus represents a rapid and robust test system with which to assess whether the glycoprotein of any newly emerging arenavirus can be efficiently processed by human SKI-1/S1P, based solely on sequence information. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Dissociation from DNA of Type III Restriction–Modification enzymes during helicase-dependent motion and following endonuclease activity

PubMed Central

Tóth, Júlia; van Aelst, Kara; Salmons, Hannah; Szczelkun, Mark D.

2012-01-01

DNA cleavage by the Type III Restriction–Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can ‘turnover’, albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. ‘DNA sliding’). PMID:22523084

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

PubMed

Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

1994-12-01

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

PubMed

Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

2014-05-01

The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

A Python Analytical Pipeline to Identify Prohormone Precursors and Predict Prohormone Cleavage Sites

PubMed Central

Southey, Bruce R.; Sweedler, Jonathan V.; Rodriguez-Zas, Sandra L.

2008-01-01

Neuropeptides and hormones are signaling molecules that support cell–cell communication in the central nervous system. Experimentally characterizing neuropeptides requires significant efforts because of the complex and variable processing of prohormone precursor proteins into neuropeptides and hormones. We demonstrate the power and flexibility of the Python language to develop components of an bioinformatic analytical pipeline to identify precursors from genomic data and to predict cleavage as these precursors are en route to the final bioactive peptides. We identified 75 precursors in the rhesus genome, predicted cleavage sites using support vector machines and compared the rhesus predictions to putative assignments based on homology to human sequences. The correct classification rate of cleavage using the support vector machines was over 97% for both human and rhesus data sets. The functionality of Python has been important to develop and maintain NeuroPred (http://neuroproteomics.scs.uiuc.edu/neuropred.html), a user-centered web application for the neuroscience community that provides cleavage site prediction from a wide range of models, precision and accuracy statistics, post-translational modifications, and the molecular mass of potential peptides. The combined results illustrate the suitability of the Python language to implement an all-inclusive bioinformatics approach to predict neuropeptides that encompasses a large number of interdependent steps, from scanning genomes for precursor genes to identification of potential bioactive neuropeptides. PMID:19169350

Avian Paramyxovirus Serotype-1: A Review of Disease Distribution, Clinical Symptoms, and Laboratory Diagnostics

PubMed Central

Hines, Nichole L.; Miller, Cathy L.

2012-01-01

Avian paramyxovirus serotype-1 (APMV-1) is capable of infecting a wide range of avian species leading to a broad range of clinical symptoms. Ease of transmission has allowed the virus to spread worldwide with varying degrees of virulence depending on the virus strain and host species. Classification systems have been designed to group isolates based on their genetic composition. The genetic composition of the fusion gene cleavage site plays an important role in virulence. Presence of multiple basic amino acids at the cleavage site allows enzymatic cleavage of the fusion protein enabling virulent viruses to spread systemically. Diagnostic tests, including virus isolation, real-time reverse-transcription PCR, and sequencing, are used to characterize the virus and identify virulent strains. Genetic diversity within APMV-1 demonstrates the need for continual monitoring for changes that may arise requiring modifications to the molecular assays to maintain their usefulness for diagnostic testing. PMID:22577610

Protease-dependent hemagglutinin cleavage contributes to alteration in chicken hemagglutination by the H3N2 influenza A virus.

PubMed

Yamaoka, Masaoki; Makino, Akiko; Sasahara, Kenji; Nastri, Aldise Mareta; Krisna, Luh Ade Wilan; Purhito, Edith Frederika; Poetranto, Emmanuel Djoko; Wulandari, Laksmi; Yudhawati, Resti; Setiawati, Landia; Setyoningrum, Retno Asih; Shinya, Kyoko

2013-01-01

The human influenza A virus (H3N2) has been the predominant influenza strain since 1992, and one property of this virus is non-agglutination of chicken erythrocytes [Ch(-) virus]. The Ch(-) virus in our study was able to acquire chicken hemagglutination [Ch(+)] by trypsin passage but not by chymotrypsin passage. Moreover, the trypsin-passaged Ch(+) viruses reacquired the Ch(-) property after a further chymotrypsin passage. In particular, genetic analysis showed no evidence of mutations in the hemagglutinin (HA) gene during either trypsin or chymotrypsin passages: the only differences found were in the HA cleavage sites between the trypsin-passaged virus and the chymotrypsin-passaged virus as determined by the N-terminal amino acid sequence. These results suggested that protease-dependent differences at the viral HA cleavage site, rather than genetic mutations, are likely to have a significant effect on the viral ability to produce chicken hemagglutination.

Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

PubMed

Chowrira, B M; Burke, J M

1991-09-03

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.

TALE-PvuII fusion proteins--novel tools for gene targeting.

PubMed

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon

PubMed Central

2013-01-01

Background The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon. Results B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. Conclusions B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants. PMID:24367943

Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase.

PubMed

Park, Jung-Ho; Yamaguchi, Yoshihiro; Inouye, Masayori

2011-08-04

MazF is an mRNA interferase which cleaves mRNAs at a specific sequence. Here, we show that in contrast to MazF-ec from Escherichia coli, which specifically cleaves ACA sequences, MazF-bs from Bacillus subtilis is an mRNA interferase that specifically cleaves a five-base sequence, UACAU. MazF homologues widely prevailing in Gram-positive bacteria were found to be highly homologous to MazF-bs, suggesting that they may also have similar cleavage specificity. This cleavage site is over-represented in the B. subtilis genes associated with biosynthesis of secondary metabolites, suggesting that MazF-bs may be involved in the regulation of the production of secondary metabolites. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Complete sequence of the genome of avian paramyxovirus type 9 and comparison with other paramyxoviruses

PubMed Central

Samuel, Arthur S.; Kumar, Sachin; Madhuri, Subbiah; Collins, Peter L.; Samal, Siba K.

2009-01-01

The complete genome consensus sequence was determined for avian paramyxovirus (APMV) serotype 9 prototype strain PMV-9/domestic Duck/New York/22/78. The genome is 15,438 nucleotides (nt) long and encodes six non-overlapping genes in the order of 3′-N-P/V/W-M-F-HN-L-5′ with intergenic regions of 0–30 nt. The genome length follows the “rule of six” and contains a 55-nt leader sequence at the 3′ end and a 47-nt trailer sequence at the 5′ end. The cleavage site of the F protein is I-R-E-G-R-I↓F, which does not conform to the conventional cleavage site of the ubiquitous cellular protease furin. The virus required exogenous protease for in vitro replication and grew only in a few established cell lines, indicating a restricted host range. Alignment and phylogenetic analysis of the predicted amino acid sequences of APMV-9 proteins with the cognate proteins of viruses of all five genera of family Paramyxoviridae showed that APMV-9 is more closely related to APMV-1 than to other APMVs. The mean death time in embryonated chicken eggs was found to be more than 120 h, indicating APMV-9 to be avirulent for chickens. PMID:19185593

APASdb: a database describing alternative poly(A) sites and selection of heterogeneous cleavage sites downstream of poly(A) signals

PubMed Central

You, Leiming; Wu, Jiexin; Feng, Yuchao; Fu, Yonggui; Guo, Yanan; Long, Liyuan; Zhang, Hui; Luan, Yijie; Tian, Peng; Chen, Liangfu; Huang, Guangrui; Huang, Shengfeng; Li, Yuxin; Li, Jie; Chen, Chengyong; Zhang, Yaqing; Chen, Shangwu; Xu, Anlong

2015-01-01

Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3′-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3′-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3′-untranslated regions (3′-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish. PMID:25378337

Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

PubMed Central

Josephs, Eric A.; Kocak, D. Dewran; Fitzgibbon, Christopher J.; McMenemy, Joshua; Gersbach, Charles A.; Marszalek, Piotr E.

2015-01-01

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex. PMID:26384421

A novel Death Defying Domain in Met entraps the active site of Caspase-3 and blocks apoptosis in hepatocytes

PubMed Central

Ma, Jihong; Zou, Chunbin; Guo, Lida; Seneviratne, Danushka S.; Tan, Xinping; Kwon, Yong-Kook; An, Jiyan; Bowser, Robert; DeFrances, Marie C.; Zarnegar, Reza

2013-01-01

Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF) is known to function as a potent anti-apoptotic mediator in normal and neoplastic cells. Herein we report that intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of Caspase-3 cleavage sites, which bait, trap and disable the active site of Caspase-3, thereby blocking the execution of apoptosis. We call this Caspase-3 cleavage motif the ‘Death Defying Domain’ (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by Caspase-3, the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of Caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of Caspase-3 activity. By gain and loss-of-function studies using restoration of DDD expression in DDD deficient hepatocytic cells, we found that both Caspase-3 sites in DDD are necessary for inhibition of Caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met’s enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of Caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion Our findings show that Met can directly inhibit Caspase-3 via a novel mechanism and promote hepato-cyte survival. Results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play. PMID:24122846

Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

PubMed Central

Narula, Gagandeep; Tse-Dinh, Yuk-Ching

2012-01-01

Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the −4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the −4 and the −5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the −4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the −4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein–DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway. PMID:22833607

Replacement of RNA hairpins by in vitro selected tetranucleotides.

PubMed Central

Dichtl, B; Pan, T; DiRenzo, A B; Uhlenbeck, O C

1993-01-01

An in vitro selection method based on the autolytic cleavage of yeast tRNA(Phe) by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucleotide sequences allow proper folding of the rest of the tRNA molecule, whereas others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNA(Phe) and the anticodon hairpin of E.coli tRNA(Phe) without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally nonessential hairpins in any RNA. Images PMID:7680121

Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection.

PubMed

Wang, Xingyu; Li, Can; Gao, Xiaomeng; Wang, Jing; Liang, Xingguo

2015-01-13

A facile and robust RNA preparation protocol was developed by combining rolling circle transcription (RCT) with RNA cleavage by RNase H. Circular DNA with a complementary sequence was used as the template for promoter-free transcription. With the aid of a 2'-O-methylated DNA, the RCT-generated tandem repeats of the desired RNA sequence were disconnected at the exact end-to-end position to harvest the desired RNA oligomers. Compared with the template DNA, more than 4 × 10(3) times the amount of small RNA products were obtained when modest cleavage was carried out during transcription. Large amounts of RNA oligomers could easily be obtained by simply increasing the reaction volume.

Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus

PubMed Central

Wang, Yupeng; Khan, Iram F.; Boissel, Sandrine; Jarjour, Jordan; Pangallo, Joseph; Thyme, Summer; Baker, David; Scharenberg, Andrew M.; Rawlings, David J.

2014-01-01

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20–22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)—a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering. PMID:24682825

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

PubMed

Mekkat, Arya; Poppleton, Erik; An, Bo; Visse, Robert; Nagase, Hideaki; Kaplan, David L; Brodsky, Barbara; Lin, Yu-Shan

2018-05-12

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage. Copyright © 2018. Published by Elsevier Inc.

Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

DOE Office of Scientific and Technical Information (OSTI.GOV)

Selmer, B.L.; Hanecak, R.; Anderson, C.W.

1981-01-01

Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, ismore » produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.« less

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

PubMed

Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki

2017-09-02

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells.

PubMed

Clevenger, Tracy N; Luna, Gabriel; Boctor, Daniel; Fisher, Steven K; Clegg, Dennis O

2016-01-01

One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg-Gly-Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg-Gly-Asp-matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg-Gly-Asp-matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration.

Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells

PubMed Central

Clevenger, Tracy N; Luna, Gabriel; Boctor, Daniel; Fisher, Steven K; Clegg, Dennis O

2016-01-01

One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg–Gly–Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration. PMID:27733898

The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

PubMed

Jasin, Maria; Haber, James E

2016-08-01

DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. Copyright © 2016. Published by Elsevier B.V.

The Democratization of Gene Editing: Insights from site-specific cleavage and double-strand break repair

PubMed Central

Jasin, Maria; Haber, James E.

2017-01-01

DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occur by homologous recombination that relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution. PMID:27261202

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem.

PubMed Central

Ananvoranich, S; Lafontaine, D A; Perreault, J P

1999-01-01

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway. PMID:10037808

Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate.

PubMed

Chang, J Y

1985-09-02

alpha-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of alpha-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by alpha-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit alpha-thrombin in 20 microliters of 50 mM ammonium bicarbonate at 25 degrees C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for alpha-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1', P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by alpha-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

PubMed Central

Lountos, George T; Austin, Brian P; Nallamsetty, Sreedevi; Waugh, David S

2009-01-01

Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single-wavelength anomolous dispersion and refined with X-ray diffraction data extending up to atomic resolution (1.13 Å). These crystallographic studies provide structural insights into the conformational changes induced upon auto-cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263-P264 peptide bond cleavage is found on a β-turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N-terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto-cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU. PMID:19165725

Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

PubMed

Luo, Ming; Gilbert, Brian; Ayliffe, Michael

2016-07-01

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs

PubMed Central

Ryan, Daniel E; Taussig, David; Steinfeld, Israel; Phadnis, Smruti M; Lunstad, Benjamin D; Singh, Madhurima; Vuong, Xuan; Okochi, Kenji D; McCaffrey, Ryan; Olesiak, Magdalena; Roy, Subhadeep; Yung, Chong Wing; Curry, Bo; Sampson, Jeffrey R; Dellinger, Douglas J

2018-01-01

Abstract CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence (‘guide sequence’) and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications. PMID:29216382

TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

PubMed Central

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

PubMed

Arlaud, G J; Gagnon, J; Porter, R R

1982-01-01

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.

1,4-Benzoquinone reductase from Phanerochaete chrysosporium: cDNA cloning and regulation of expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akileswaran, L.; Brock, B.J.; Cereghino, J.L.

1999-02-01

A cDNA clone encoding a quinone reductase (QR) from the white rot basidiomycete Phanerochaete chrysosporium was isolated and sequenced. The cDNA consisted of 1,007 nucleotides and a poly(A) tail and encoded a deduced protein containing 271 amino acids. The experimentally determined eight-amino-acid N-germinal sequence of the purified QR protein from P. chrysosporium matched amino acids 72 to 79 of the predicted translation product of the cDNA. The M{sub r} of the predicted translation product, beginning with Pro-72, was essentially identical to the experimentally determined M{sub r} of one monomer of the QR dimer, and this finding suggested that QR ismore » synthesized as a proenzyme. The results of in vitro transcription-translation experiments suggested that QR is synthesized as a proenzyme with a 71-amino-acid leader sequence. This leader sequence contains two potential KEX2 cleavage sites and numerous potential cleavage sites for dipeptidyl aminopeptidase. The QR activity in cultures of P. chrysosporium increased following the addition of 2-dimethoxybenzoquinone, vanillic acid, or several other aromatic compounds. An immunoblot analysis indicated that induction resulted in an increase in the amount of QR protein, and a Northern blot analysis indicated that this regulation occurs at the level of the qr mRNA.« less

DeepSig: deep learning improves signal peptide detection in proteins.

PubMed

Savojardo, Castrense; Martelli, Pier Luigi; Fariselli, Piero; Casadio, Rita

2018-05-15

The identification of signal peptides in protein sequences is an important step toward protein localization and function characterization. Here, we present DeepSig, an improved approach for signal peptide detection and cleavage-site prediction based on deep learning methods. Comparative benchmarks performed on an updated independent dataset of proteins show that DeepSig is the current best performing method, scoring better than other available state-of-the-art approaches on both signal peptide detection and precise cleavage-site identification. DeepSig is available as both standalone program and web server at https://deepsig.biocomp.unibo.it. All datasets used in this study can be obtained from the same website. pierluigi.martelli@unibo.it. Supplementary data are available at Bioinformatics online.

Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

PubMed Central

Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya

2016-01-01

Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253

Genetic Diversity of Avian Paramyxovirus Type 6 Isolated from Wild Ducks in the Republic of Korea.

PubMed

Choi, Kang-Seuk; Kim, Ji-Ye; Lee, Hyun-Jeong; Jang, Min-Jun; Kwon, Hyuk-Moo; Sung, Haan-Woo

2018-03-08

Eleven avian paramyxovirus type 6 (APMV-6) isolates from Eurasian Wigeon ( n=5; Anas penelope), Mallards ( n=2; Anas platyrhynchos), and unknown species of wild ducks ( n=4) from Korea were analyzed based on the nucleotide (nt) and deduced amino acid (aa) sequences of the fusion (F) gene. Fecal samples were collected in 2010-2014. Genotypes were assigned based on phylogenetic analyses. Our results revealed that APMV-6 could be classified into at least two distinct genotypes, G1 and G2. The open reading frame (ORF) of the G1 genotype was 1,668 nt in length, and the putative F0 cleavage site sequence was 113 PAPEPRL 119 . The G2 genotype viruses included five isolates from Eurasian wigeons and four isolates from unknown waterfowl species, together with two reference APMV-6 strains from the Red-necked Stint ( Calidris ruficollis) from Japan and an unknown duck from Italy. There was an N-truncated ORF (1,638 nt), due to an N-terminal truncation of 30 nt in the signal peptide region of the F gene, and the putative F0 cleavage site sequence was 103 SIREPRL 109 . The genetic diversity and ecology of APMV-6 are discussed.

Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB

PubMed Central

Fröbel, Julia; Rose, Patrick; Lausberg, Frank; Blümmel, Anne-Sophie; Freudl, Roland; Müller, Matthias

2012-01-01

The twin-arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates the transmembrane transport of folded proteins, which harbour signal sequences with a conserved twin-arginine motif. Many Tat translocases comprise the three membrane proteins TatA, TatB and TatC. TatC was previously shown to be involved in recognizing twin-arginine signal peptides. Here we show that beyond recognition, TatC mediates the transmembrane insertion of a twin-arginine signal sequence, thereby translocating the signal sequence cleavage site across the bilayer. In the absence of TatB, this can lead to the removal of the signal sequence even from a translocation-incompetent substrate. Hence interaction of twin-arginine signal peptides with TatB counteracts their premature cleavage uncoupled from translocation. This capacity of TatB is not shared by the homologous TatA protein. Collectively our results suggest that TatC is an insertase for twin-arginine signal peptides and that translocation-proficient signal sequence recognition requires the concerted action of TatC and TatB. PMID:23250441

Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB.

PubMed

Fröbel, Julia; Rose, Patrick; Lausberg, Frank; Blümmel, Anne-Sophie; Freudl, Roland; Müller, Matthias

2012-01-01

The twin-arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates the transmembrane transport of folded proteins, which harbour signal sequences with a conserved twin-arginine motif. Many Tat translocases comprise the three membrane proteins TatA, TatB and TatC. TatC was previously shown to be involved in recognizing twin-arginine signal peptides. Here we show that beyond recognition, TatC mediates the transmembrane insertion of a twin-arginine signal sequence, thereby translocating the signal sequence cleavage site across the bilayer. In the absence of TatB, this can lead to the removal of the signal sequence even from a translocation-incompetent substrate. Hence interaction of twin-arginine signal peptides with TatB counteracts their premature cleavage uncoupled from translocation. This capacity of TatB is not shared by the homologous TatA protein. Collectively our results suggest that TatC is an insertase for twin-arginine signal peptides and that translocation-proficient signal sequence recognition requires the concerted action of TatC and TatB.

Albumin Redhill (-1 Arg, 320 Ala----Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site.

PubMed

Brennan, S O; Myles, T; Peach, R J; Donaldson, D; George, P M

1990-01-01

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind 63Ni2+ and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni2+ was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation (since proalbumin contains adjacent Arg residues at -1 and -2) or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala----Thr. This introduces an Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg----Cys. This introduces a prepro sequence, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr- Ser-Arg-Gly-Val-Phe-Cys-Arg (cf.-Tyr-Ser-Arg-Gly-Val-Phe-Arg-Arg- in normal human pre-proalbumin). We propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.

Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

PubMed Central

Sharma, Kishor; Tollervey, David

1999-01-01

The loop of a stem structure close to the 5′ end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A1, the 5′ end of the 18S rRNA, and at site A2, located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3–pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5′ external transcribed spacer (5′ ETS). Cleavage at site A0 in the yeast 5′ ETS strictly requires base pairing between U3 and a sequence within the 5′ ETS. In contrast, the U3-18S interaction is not required for A0 cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A1 cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A1 cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing. PMID:10454548

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

PubMed

Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

2008-04-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

PubMed Central

Wang, Chao; Lv, Yangyong; Wang, Bin; Yin, Chao; Lin, Ying; Pan, Li

2015-01-01

The genome-scale delineation of in vivo protein–DNA interactions is key to understanding genome function. Only ∼5% of transcription factors (TFs) in the Aspergillus genus have been identified using traditional methods. Although the Aspergillus oryzae genome contains >600 TFs, knowledge of the in vivo genome-wide TF-binding sites (TFBSs) in aspergilli remains limited because of the lack of high-quality antibodies. We investigated the landscape of in vivo protein–DNA interactions across the A. oryzae genome through coupling the DNase I digestion of intact nuclei with massively parallel sequencing and the analysis of cleavage patterns in protein–DNA interactions at single-nucleotide resolution. The resulting map identified overrepresented de novo TF-binding motifs from genomic footprints, and provided the detailed chromatin remodeling patterns and the distribution of digital footprints near transcription start sites. The TFBSs of 19 known Aspergillus TFs were also identified based on DNase I digestion data surrounding potential binding sites in conjunction with TF binding specificity information. We observed that the cleavage patterns of TFBSs were dependent on the orientation of TF motifs and independent of strand orientation, consistent with the DNA shape features of binding motifs with flanking sequences. PMID:25883143

Characterization of molecular interactions between Zika virus protease and peptides derived from the C-terminus of NS2B.

PubMed

Li, Yan; Loh, Ying Ru; Hung, Alvin W; Kang, CongBao

2018-06-21

Zika virus (ZIKV) protease is a two-component complex in which NS3 contains the catalytic triad and NS2B cofactor region is important for protease folding and activity. A protease construct-eZiPro without the transmembrane domains of NS2B was designed. Structural study on eZiPro reveals that the Thr-Gly-Lys-Arg (TGKR) sequence at the C-terminus of NS2B binds to the active site after cleavage. The bZiPro construct only contains NS2B cofactor region and the N-terminus of NS3 without any artificial linker or protease cleavage site, giving rise to an empty pocket accessible to substrate and inhibitor binding. Herein, we demonstrate that the TGKR sequence of NS2B in eZiPro is dynamic. Peptides from NS2B with various lengths exhibit different binding affinities to bZiPro. TGKR binding to the active site in eZiPro does not affect protease binding to small-molecule compounds. Our results suggest that eZiPro will also be useful for evaluating small-molecule protease inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

RNA-guided genome editing for target gene mutations in wheat.

PubMed

Upadhyay, Santosh Kumar; Kumar, Jitesh; Alok, Anshu; Tuli, Rakesh

2013-12-09

The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder.

PubMed

Conlon, J M; Davis, M S; Falkmer, S; Thim, L

1987-11-02

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.

5-Nitrosalicylic Acid as a Novel Matrix for In-Source Decay in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

PubMed Central

Asakawa, Daiki

2013-01-01

The matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) of peptides and glycans was studied using an oxidizing chemical, 5-nitrosalicylic acid (5-NSA) as the matrix. The use of 5-NSA for the MALDI-ISD of peptides and glycans promoted fragmentation pathways involving “hydrogen-deficient” radical precursors. Hydrogen abstraction from peptides resulted in the production of a “hydrogen-deficient” peptide radical that contained a radical site on the amide nitrogen in the peptide backbone with subsequent radical-induced cleavage at the Cα–C bonds. Cleavage at the Cα–C bond leads to the production of an a•/x fragment pair and the radical a• ions then undergo further hydrogen abstraction to form a ions after Cα–C bond cleavage. Since the Pro residue does not contain a nitrogen-centered radical site, Cα–C bond cleavage does not occur at this site. Alternatively, the specific cleavage of CO−N bonds leads to a b•/y fragment pair at Xxx−Pro which occurs via hydrogen abstraction from the Cα−H in the Pro residue. In contrast, “hydrogen-deficient” glycan radicals were generated by hydrogen abstraction from hydroxyl groups in glycans. Both glycosidic and cross-ring cleavages occurred as the result of the degradation of “hydrogen-deficient” glycan radicals. Cross-ring cleavage ions are potentially useful in linkage analysis, one of the most critical steps in the characterization of glycans. Moreover, isobaric glycans could be distinguished by structure specific ISD ions, and the molar ratio of glycan isomers in a mixture can be estimated from their fragment ions abundance ratios. MALDI-ISD with 5-NSA could be a useful method for the sequencing of peptides including the location of post-translational modifications, identification and semi-quantitative analysis of mixtures of glycan isomers. PMID:24860709

PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation

PubMed Central

Yang, Yan; Li, Wencheng; Hoque, Mainul; Hou, Liming; Shen, Steven; Tian, Bin; Dynlacht, Brian D.

2016-01-01

The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and RNA polymerase II (PolII) elongation. Here, we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, we identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3’ ends of transcripts. Moreover, we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation, although there was little impact of Paf1C depletion on other histone modifications, including tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site (TSS)-proximal pA sites and regulating pA site choice in 3’UTRs. PMID:26765774

Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

PubMed

Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

2017-01-01

Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto

PubMed Central

Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

2017-01-01

Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3’UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3’UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3’UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo. PMID:28253363

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

PubMed Central

2011-01-01

Background Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. Results An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). Conclusions The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy. PMID:21864341

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.

PubMed

Sobolewski, Ireneusz; Polska, Katarzyna; Zylicz-Stachula, Agnieszka; Jeżewska-Frąckowiak, Joanna; Rak, Janusz; Skowron, Piotr

2011-08-24

Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

PubMed

Pauly, Thorsten; Ratliff, Miriam; Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

2008-07-16

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

PubMed Central

Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

2008-01-01

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS. PMID:18629001

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Yuqian; Hellinga, Homme W.; Beese, Lorena S.

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'–3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed endsmore » at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.« less

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I.

PubMed

Shi, Yuqian; Hellinga, Homme W; Beese, Lorena S

2017-06-06

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.

Multifaceted regulation of V(D)J recombination

NASA Astrophysics Data System (ADS)

Wang, Guannan

V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg 2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.

Sequence specificity of the hammerhead ribozyme revisited; the NHH rule.

PubMed Central

Kore, A R; Vaish, N K; Kutzke, U; Eckstein, F

1998-01-01

The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH. PMID:9722629

DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion

PubMed Central

Schwarz, Friedrich W.; van Aelst, Kara; Tóth, Júlia; Seidel, Ralf; Szczelkun, Mark D.

2011-01-01

DNA cleavage by the Type III Restriction–Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision. PMID:21724613

Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA

NASA Astrophysics Data System (ADS)

Schmidt, Thomas P.; Perna, Anna M.; Fugmann, Tim; Böhm, Manja; Jan Hiss; Haller, Sarah; Götz, Camilla; Tegtmeyer, Nicole; Hoy, Benjamin; Rau, Tilman T.; Neri, Dario; Backert, Steffen; Schneider, Gisbert; Wessler, Silja

2016-03-01

The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.

Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes.

PubMed

Biedka, Stephanie; Micic, Jelena; Wilson, Daniel; Brown, Hailey; Diorio-Toth, Luke; Woolford, John L

2018-04-24

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C 2 site of 27SB pre-rRNA. C 2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C 2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C 2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C 2 cleavage. Interestingly, when C 2 cleavage is directly blocked by depleting or inactivating the C 2 endonuclease, assembly progresses through all other subsequent steps. © 2018 Biedka et al.

Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA.

PubMed Central

Hendrix, C; Anné, J; Joris, B; Van Aerschot, A; Herdewijn, P

1996-01-01

Four GUC triplets in the coding region of the MRNA of interleukin 6 (IL-6) were examined for their suitabilty to serve as a target for hammerhead ribozome-mediated cleavage. This selection procedure was performed with the intention to downregulate IL-6 production as a potential treatment of those diseases in which IL-6 overexpression is involved. Hammerhead ribozymes and their respective short synthetic substrates (19-mers) were synthesized for these four GUC triplets. Notwithstanding the identical catalytic core sequences, the difference in base composition of the helices involved in substrate binding caused substantial variation in cleavage activity. The cleavage reactions on the 1035 nucleotide IL-6 mRNA transcript revealed that two ribozymes were able to cleave this substrate, showing a decrease in catalytic efficiency to 1/30 and 1/300 of the short substrate. This study indicates that the GUC triplet located at nucleotide 510 of the mRNA of IL-6 is the best site for hammerhead ribozyme-mediated cleavage. We suggest that in future targeting of chemically modified hammerhead ribosomes for cleavage of IL-6 RNA should be directed at this location. PMID:8670082

Newcastle disease virus fusion and haemagglutinin-neuraminidase proteins contribute to its macrophage host range

USDA-ARS?s Scientific Manuscript database

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic damage to international poultry industry. Different strains of NDV express a wide range of virulence that is primarily dependent on the amino acid sequence of the strain’s fusion (F) protein cleavage site. Two c...

Novel Variant of Tickborne Encephalitis Virus, Russia

PubMed Central

Ternovoi, Vladimir A.; Protopopova, Elena V.; Chausov, Eugene V.; Novikov, Dmitry V.; Leonova, Galina N.; Netesov, Sergey V.

2007-01-01

We isolated a novel strain of tickborne encephalitis virus (TBEV), Glubinnoe/2004, from a patient with a fatal case in Russia. We sequenced the strain, whose landmark features included 57 amino acid substitutions and 5 modified cleavage sites. Phylogenetically, Glubinnoe/2004 is a novel variant that belongs to the Eastern type of TBEV. PMID:18258012

Enzyme-triggered Gelation: Targeting Proteases with Internal Cleavage Sites

PubMed Central

Bremmer, Steven C.

2014-01-01

A generalizable method for detecting protease activity via gelation is described. A recognition sequence is used to target the protease of interest while a second protease is used to remove the residual residues from the gelator scaffold. Using this approach, selective assays for both MMP-9 and PSA are demonstrated. PMID:24394494

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

PubMed

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

NASA Astrophysics Data System (ADS)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

E2F mediates enhanced alternative polyadenylation in proliferation.

PubMed

Elkon, Ran; Drost, Jarno; van Haaften, Gijs; Jenal, Mathias; Schrier, Mariette; Oude Vrielink, Joachim A F; Agami, Reuven

2012-07-02

The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.

Analysis of alterative cleavage and polyadenylation by 3′ region extraction and deep sequencing

PubMed Central

Hoque, Mainul; Ji, Zhe; Zheng, Dinghai; Luo, Wenting; Li, Wencheng; You, Bei; Park, Ji Yeon; Yehia, Ghassan; Tian, Bin

2012-01-01

Alternative cleavage and polyadenylation (APA) leads to mRNA isoforms with different coding sequences (CDS) and/or 3′ untranslated regions (3′UTRs). Using 3′ Region Extraction And Deep Sequencing (3′READS), a method which addresses the internal priming and oligo(A) tail issues that commonly plague polyA site (pA) identification, we comprehensively mapped pAs in the mouse genome, thoroughly annotating 3′ ends of genes and revealing over five thousand pAs (~8% of total) flanked by A-rich sequences, which have hitherto been overlooked. About 79% of mRNA genes and 66% of long non-coding RNA (lncRNA) genes have APA; but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Promoter-distal pAs become relatively more abundant during embryonic development and cell differentiation, a trend affecting pAs in both 3′-most exons and upstream regions. Upregulated isoforms generally have stronger pAs, suggesting global modulation of the 3′ end processing activity in development and differentiation. PMID:23241633

Concurrent Automated Sequencing of the Glycan and Peptide Portions of O-Linked Glycopeptide Anions by Ultraviolet Photodissociation Mass Spectrometry

PubMed Central

Madsen, James A.; Ko, Byoung Joon; Xu, Hua; Iwashkiw, Jeremy A.; Robotham, Scott A.; Shaw, Jared B.; Feldman, Mario F.; Brodbelt, Jennifer S.

2013-01-01

O -glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry (MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly- and multiply-deprotonated glycopeptides from the model glycoprotein kappa casein, resulting in production of both peptide and glycan product ions that afforded 100% sequence coverage of the peptide and glycan moieties from a single MS/MS event. The most abundant and frequent peptide sequence ions were a/x-type products, which, importantly, were found to retain the labile glycan modifications. The glycan-specific ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions) in addition to some less common cross-ring cleavages. Based on the UVPD fragmentation patterns, an automated database searching strategy (based on the MassMatrix algorithm) was designed that is specific for the analysis of glycopeptide anions by UVPD. This algorithm was used to identify glycopeptides from mixtures of glycosylated and non-glycosylated peptides, sequence both glycan and peptide moieties simultaneously, and pinpoint the correct site(s) of glycosylation. This methodology was applied to uncover novel site-specificity of the O-linked glycosylated OmpA/MotB from the “superbug” A. baumannii to help aid in the elucidation of the functional role that protein glycosylation plays in pathogenesis. PMID:24006841

Analysis of apolipoprotein A-I as a substrate for matrix metalloproteinase-14

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jun Hyoung; Park, Sung-Min; Park, Ki-Hoon

2011-05-27

Highlights: {yields} MMP-14 degrades apoA-I more efficiently than other tested MMPs. {yields} Lipid-free apoA-I is more susceptible to MMPs than lipid-bound apoA-I. {yields} MMP-14 cleavage sites on apoA-I have been determined. {yields} Cleavage of apoA-I by MMP-14 impairs its ability to form HDL. -- Abstract: Substrates for matrix metalloproteinase (MMP)-14 were previously identified in human plasma using proteomic techniques. One putative MMP-14 substrate was apolipoprotein A-I (apoA-I), a major component of high-density lipoprotein (HDL). In vitro cleavage assays showed that lipid-free apoA-I is a more accessible substrate for MMP-14 compared to lipid-bound apoA-I, and that MMP-14 is more prone tomore » digest apoA-I than MMP-3. The 28-kDa apoA-I was cleaved into smaller fragments of 27, 26, 25, 22, and 14-kDa by MMP-14. ApoA-I sites cleaved by MMP-14 were determined by isotope labeling of C-termini derived from the cleavage and analysis of the labeled peptides by mass spectrometry, along with N-terminal sequencing of the fragments. Cleavage of apoA-I by MMP-14 resulted in a loss of ability to form HDL. Our results suggest that cleavage of lipid-free apoA-I by MMP-14 may contribute to reduced HDL formation, and this may be occurring during the development of various vascular diseases as lipid metabolism is disrupted.« less

Delta ribozyme has the ability to cleave in transan mRNA.

PubMed Central

Roy, G; Ananvoranich, S; Perreault, J P

1999-01-01

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy. PMID:9927724

The structure of a thermophilic kinase shapes fitness upon random circular permutation

PubMed Central

Jones, Alicia M.; Mehta, Manan M.; Thomas, Emily E.; Atkinson, Joshua T.; Segall-Shapiro, Thomas H.; Liu, Shirley; Silberg, Jonathan J.

2016-01-01

Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement where native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein’s functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AK with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and they reveal a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection. PMID:26976658

The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation.

PubMed

Jones, Alicia M; Mehta, Manan M; Thomas, Emily E; Atkinson, Joshua T; Segall-Shapiro, Thomas H; Liu, Shirley; Silberg, Jonathan J

2016-05-20

Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement in which native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein's functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AKs with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and it reveals a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection.

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage

PubMed Central

Brok-Volchanskaya, Vera S.; Kadyrov, Farid A.; Sivogrivov, Dmitry E.; Kolosov, Peter M.; Sokolov, Andrey S.; Shlyapnikov, Michael G.; Kryukov, Valentine M.; Granovsky, Igor E.

2008-01-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3′ 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TψC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages. PMID:18281701

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yanli; Sheng, Gang; Juranek, Stefan

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas twomore » critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.« less

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

PubMed Central

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Cleavage site and Ectodomain of HA2 sub-unit sequence of three equine influenza virus isolated in Morocco

PubMed Central

2014-01-01

Background The equine influenza (EI) is an infectious and contagious disease of the upper respiratory tract of horses. Two outbreaks were notified in Morocco during 1997 and 2004 respectively in Nador and Essaouira. The aims of the present study concern the amino acids sequences comparison with reference strain A/equine/Miami/1963(H3N8) of the HA2 subunit including the cleavage site of three equine influenza viruses (H3N8) isolated in Morocco: A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004 (H3N8) and A/equine/Essaouira/3/2004 (H3N8). Results The obtained results demonstrated that the substitutions were located at Ectodomain (ED) and transmembrane domain (TD), and they have only one arginine in cleavage site (HA1-PEKQI-R329-GI-HA2). In the Ectodomain, the mutation N/154 2 /T deleted the NGT glycosylation site at position 154 for both strains A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8). Except for mutation D/1602/Y of the A/equine/Nador/1/1997(H3N8) strain, the other mutations were involved in non conserved sites. While the transmembrane domain (TM) of the strain A/equine/Essaouira/3/2004(H3N8) exhibits a substitution at residue C/199 2 /F. For the A/equine/Nador/1/1997(H3N8) strain the HA2 shows a mutation at residue M/207 2 /L. Three Moroccan strains reveals a common substitution at the residue E/211 2 /Q located between transmembrane domain TM and the cytoplasmic domain (CD). Conclusion The given nature virulence of three Moroccan strains, the identified and reported mutations certainly played a permissive role of infection viral process. PMID:25016480

A comprehensive analysis of 3′ end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation

PubMed Central

Gruber, Andreas J.; Schmidt, Ralf; Gruber, Andreas R.; Martin, Georges; Ghosh, Souvik; Belmadani, Manuel; Keller, Walter

2016-01-01

Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3′ untranslated region (3′ UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3′ end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3′ UTR lengths remain to be characterized. Through a uniform analysis of a large number of 3′ end sequencing data sets, we have uncovered 18 signals, six of which are novel, whose positioning with respect to pre-mRNA cleavage sites indicates a role in pre-mRNA 3′ end processing in both mouse and human. With 3′ end sequencing we have demonstrated that the heterogeneous ribonucleoprotein C (HNRNPC), which binds the poly(U) motif whose frequency also peaks in the vicinity of polyadenylation (poly(A)) sites, has a genome-wide effect on poly(A) site usage. HNRNPC-regulated 3′ UTRs are enriched in ELAV-like RBP 1 (ELAVL1) binding sites and include those of the CD47 gene, which participate in the recently discovered mechanism of 3′ UTR–dependent protein localization (UDPL). Our study thus establishes an up-to-date, high-confidence catalog of 3′ end processing sites and poly(A) signals, and it uncovers an important role of HNRNPC in regulating 3′ end processing. It further suggests that U-rich elements mediate interactions with multiple RBPs that regulate different stages in a transcript's life cycle. PMID:27382025

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

PubMed

Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

2018-02-27

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

Structure and dynamics of mesophilic variants from the homing endonuclease I-DmoI

NASA Astrophysics Data System (ADS)

Alba, Josephine; Marcaida, Maria Jose; Prieto, Jesus; Montoya, Guillermo; Molina, Rafael; D'Abramo, Marco

2017-12-01

I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity.

Mucin (MUC1) Expression and Function in Prostate Cancer Cells

DTIC Science & Technology

2004-03-01

Biophysical Research Communications 293 (2002) 1183-1190 1189 site, and is a feature of MUC4 metabolism [36]. [12] S. Zotter, P.C. Hageman, A. Lossnitzer...W.J. Mooi, J. Hilgers, Although the cleavage site sequence in MUC4 differs Tissue and tumor distribution of human polymorphic epithelial from that of...family of putative secreted (2001) 715-720. and membrane-associated MUC4 mucins, Eur. J. Biochem. 267 [261 M. Boshell, E-N. Lalani, L. Pemberton, J

Effects of RFRP2 and RF9 on secretion of luteinizing hormone in prepubertal gilts

USDA-ARS?s Scientific Manuscript database

In most mammals, the RF-amide related peptide (RFRP) pre-pro-protein contains cleavage sites for 2 peptides (RFRP1 and RFRP3). Our laboratory did not observe RFRP3-induced suppression of LH secretion in gilts. However, the porcine sequence encodes an additional peptide (RFRP2) with an amidated C-ter...

GPS-CCD: A Novel Computational Program for the Prediction of Calpain Cleavage Sites

PubMed Central

Gao, Xinjiao; Ma, Qian; Ren, Jian; Xue, Yu

2011-01-01

As one of the most essential post-translational modifications (PTMs) of proteins, proteolysis, especially calpain-mediated cleavage, plays an important role in many biological processes, including cell death/apoptosis, cytoskeletal remodeling, and the cell cycle. Experimental identification of calpain targets with bona fide cleavage sites is fundamental for dissecting the molecular mechanisms and biological roles of calpain cleavage. In contrast to time-consuming and labor-intensive experimental approaches, computational prediction of calpain cleavage sites might more cheaply and readily provide useful information for further experimental investigation. In this work, we constructed a novel software package of GPS-CCD (Calpain Cleavage Detector) for the prediction of calpain cleavage sites, with an accuracy of 89.98%, sensitivity of 60.87% and specificity of 90.07%. With this software, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. In this regard, GPS-CCD 1.0 is considered to be a useful tool for experimentalists. The online service and local packages of GPS-CCD 1.0 were implemented in JAVA and are freely available at: http://ccd.biocuckoo.org/. PMID:21533053

Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production.

PubMed

Han, Bo W; Wang, Wei; Li, Chengjian; Weng, Zhiping; Zamore, Phillip D

2015-05-15

PIWI-interacting RNAs (piRNAs) protect the animal germ line by silencing transposons. Primary piRNAs, generated from transcripts of genomic transposon "junkyards" (piRNA clusters), are amplified by the "ping-pong" pathway, yielding secondary piRNAs. We report that secondary piRNAs, bound to the PIWI protein Ago3, can initiate primary piRNA production from cleaved transposon RNAs. The first ~26 nucleotides (nt) of each cleaved RNA becomes a secondary piRNA, but the subsequent ~26 nt become the first in a series of phased primary piRNAs that bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. The ping-pong pathway increases only the abundance of piRNAs, whereas production of phased primary piRNAs from cleaved transposon RNAs adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. Copyright © 2015, American Association for the Advancement of Science.

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles.

PubMed

Nguyen, Albert T; Feasley, Christa L; Jackson, Ken W; Nitz, Theodore J; Salzwedel, Karl; Air, Gillian M; Sakalian, Michael

2011-12-07

Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

PubMed Central

Borda, Emily J.; Markley, John C.; Sigurdsson, Snorri Th.

2003-01-01

We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn2+. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes. PMID:12736309

Proton-driven amide bond-cleavage pathways of gas-phase peptide ions lacking mobile protons.

PubMed

Bythell, Benjamin J; Suhai, Sándor; Somogyi, Arpád; Paizs, Béla

2009-10-07

The mobile proton model (Dongre, A. R., Jones, J. L., Somogyi, A. and Wysocki, V. H. J. Am. Chem. Soc. 1996, 118 , 8365-8374) of peptide fragmentation states that the ionizing protons play a critical role in the gas-phase fragmentation of protonated peptides upon collision-induced dissociation (CID). The model distinguishes two classes of peptide ions, those with or without easily mobilizable protons. For the former class mild excitation leads to proton transfer reactions which populate amide nitrogen protonation sites. This enables facile amide bond cleavage and thus the formation of b and y sequence ions. In contrast, the latter class of peptide ions contains strongly basic functionalities which sequester the ionizing protons, thereby often hindering formation of sequence ions. Here we describe the proton-driven amide bond cleavages necessary to produce b and y ions from peptide ions lacking easily mobilizable protons. We show that this important class of peptide ions fragments by different means from those with easily mobilizable protons. We present three new amide bond cleavage mechanisms which involve salt-bridge, anhydride, and imine enol intermediates, respectively. All three new mechanisms are less energetically demanding than the classical oxazolone b(n)-y(m) pathway. These mechanisms offer an explanation for the formation of b and y ions from peptide ions with sequestered ionizing protons which are routinely fragmented in large-scale proteomics experiments.

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I

PubMed Central

Shi, Yuqian; Hellinga, Homme W.; Beese, Lorena S.

2017-01-01

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5′-nuclease superfamily. Its dominant, processive 5′–3′ exonuclease and secondary 5′-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5′-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5′ flaps. Their distinctive 5′ ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5′ flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity. PMID:28533382

Metastable Atom-Activated Dissociation Mass Spectrometry of Phosphorylated and Sulfonated Peptides in Negative Ion Mode

NASA Astrophysics Data System (ADS)

Cook, Shannon L.; Jackson, Glen P.

2011-06-01

The dissociation behavior of phosphorylated and sulfonated peptide anions was explored using metastable atom-activated dissociation mass spectrometry (MAD-MS) and collision-induced dissociation (CID). A beam of high kinetic energy helium (He) metastable atoms was exposed to isolated phosphorylated and sulfonated peptides in the 3- and 2- charge states. Unlike CID, where phosphate losses are dominant, the major dissociation channels observed using MAD were Cα - C peptide backbone cleavages and neutral losses of CO2, H2O, and [CO2 + H2O] from the charge reduced (oxidized) product ion, consistent with an electron detachment dissociation (EDD) mechanism such as Penning ionization. Regardless of charge state or modification, MAD provides ample backbone cleavages with little modification loss, which allows for unambiguous PTM site determination. The relative abundance of certain fragment ions in MAD is also demonstrated to be somewhat sensitive to the number and location of deprotonation sites, with backbone cleavage somewhat favored adjacent to deprotonated sites like aspartic acid residues. MAD provides a complementary dissociation technique to CID, ECD, ETD, and EDD for peptide sequencing and modification identification. MAD offers the unique ability to analyze highly acidic peptides that contain few to no basic amino acids in either negative or positive ion mode.

Influence of codon usage bias on FGLamide-allatostatin mRNA secondary structure.

PubMed

Martínez-Pérez, Francisco; Bendena, William G; Chang, Belinda S W; Tobe, Stephen S

2011-03-01

The FGLamide allatostatins (ASTs) are invertebrate neuropeptides which inhibit juvenile hormone biosynthesis in Dictyoptera and related orders. They also show myomodulatory activity. FGLamide AST nucleotide frequencies and codon bias were investigated with respect to possible effects on mRNA secondary structure. 367 putative FGLamide ASTs and their potential endoproteolytic cleavage sites were identified from 40 species of crustaceans, chelicerates and insects. Among these, 55% comprised only 11 amino acids. An FGLamide AST consensus was identified to be (X)(1→16)Y(S/A/N/G)FGLGKR, with a strong bias for the codons UUU encoding for Phe and AAA for Lys, which can form strong Watson-Crick pairing in all peptides analyzed. The physical distance between these codons favor a loop structure from Ser/Ala-Phe to Lys-Arg. Other loop and hairpin loops were also inferred from the codon frequencies in the N-terminal motif, and the first amino acids from the C-terminal motif, or the dibasic potential endoproteolytic cleavage site. Our results indicate that nucleotide frequencies and codon usage bias in FGLamide ASTs tend to favor mRNA folds in the codon sequence in the C-terminal active peptide core and at the dibasic potential endoproteolytic cleavage site. Copyright © 2010 Elsevier Inc. All rights reserved.

Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

PubMed

Olorunniji, Femi J; McPherson, Arlene L; Pavlou, Hania J; McIlwraith, Michael J; Brazier, John A; Cosstick, Richard; Stark, W Marshall

2015-07-13

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Affinity cleavage at the putative metal-binding site of pigeon liver malic enzyme by the Fe(2+)-ascorbate system.

PubMed

Wei, C H; Chou, W Y; Huang, S M; Lin, C C; Chang, G G

1994-06-28

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at neutral pH and 0 or 25 degrees C. Omitting the ascorbate or replacing the ferrous ion with manganese ion did not lead to any inactivation. Manganese, magnesium, zinc, cobalt, or calcium ion at 200 molar excess over ferrous ion offered complete protection of the enzyme from Fe(2+)-induced inactivation. Ni2+ provided partial protection, while Ba2+ or imidazole was ineffective in protection. Addition of 4 mM Mn2+ or 5 mM EDTA into a partially modified enzyme stopped further inactivation of the enzyme. Inclusion of substrates (L-malate or NADP+, singly or in combination) in the incubation mixture did not affect the inactivation rate. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native pigeon liver malic enzyme had a subunit M(r) of 65,000. The inactivated enzyme with residual activity of only 0.3% was cleaved into two fragments with M(r) of 31,000 and 34,000, respectively. The cleavage site was identified as the peptide bond between Asp258 and Ile259. Native pigeon liver malic enzyme was blocked at the N-terminus. Cleavage at the putative metal-binding site exposed a new N-terminus, which was identified to be at the 34-kDa fragment containing the C-terminal half of original sequence 259-557. Our results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at Asp258 and/or some other essential amino acid residues that caused enzyme inactivation. The modified enzyme was then affinity cleaved at the Mn(2+)-binding site.

NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization.

PubMed Central

Yu, W. F.; Tung, C. S.; Wang, H.; Tasayco, M. L.

2000-01-01

Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer. Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer. PMID:10739243

E2F mediates enhanced alternative polyadenylation in proliferation

PubMed Central

2012-01-01

Background The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Results Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Conclusions Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation. PMID:22747694

Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy.

PubMed

Shibata, Mikihiro; Nishimasu, Hiroshi; Kodera, Noriyuki; Hirano, Seiichi; Ando, Toshio; Uchihashi, Takayuki; Nureki, Osamu

2017-11-10

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.

Antibodies to H1 histone from the sera of HIV-infected patients recognize and catalyze site-specific degradation of this histone.

PubMed

Baranova, Svetlana V; Dmitrienok, Pavel S; Ivanisenko, Nikita V; Buneva, Valentina N; Nevinsky, Georgy A

2017-03-01

Histones and their posttranslational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive feature of some autoimmune and viral diseases. Electrophoretically and immunologically homogeneous IgGs containing no canonical enzymes were isolated from sera of human immunodeficiency virus-infected patients by chromatography on several affinity sorbents. In contrast to canonical proteases (trypsin, chymotrypsin, and proteinase K), IgGs from human immunodeficiency virus-infected patients purified by affinity chromatography on Sepharose containing immobilized histones specifically recognized and hydrolyzed only histones but not many other tested globular proteins. Using matrix-assisted laser desorption/ionization mass spectrometry, the sites of H1 histone (193 amino acids [AAs]) cleavage by anti-H1 histone IgGs were determined for the first time. It was shown that 1 cluster of 2 major and 4 moderate sites of cleavage is located at the beginning (106-112 AAs) of the known antigenic determinants disposed at the long C-terminal sequence of H1. Two clusters of minor and very weak sites of the protein cleavage correspond to middle (8 sites, 138-158 AAs) and terminal (5 sites, 166-176 AAs) parts of the antigenic determinants. It was shown that in contrast to canonical proteases, N-terminal part of H1 histone (1-136 AAs) containing no antigenic determinants is an unpredictably very resistant against hydrolysis by abzymes, while it can be easily cleavage by canonical proteases. Because histones act as damage-associated molecules, abzymes against H1 and other histones can play important role in pathogenesis of acquired immune deficiency syndrome and probably other different diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses.

PubMed

Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Keller, Ulrich Auf dem; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

2016-01-01

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1'. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Alternative Enzymes Lead to Improvements in Sequence Coverage and PTM Analysis

PubMed Central

Hooper, Kyle; Rosenblatt, Michael; Urh, Marjeta; Saveliev, Sergei; Hosfield, Chris; Kobs, Gary; Ford, Michael; Jones, Richard; Amunugama, Ravi; Allen, David; Brazas, Robert

2013-01-01

The profiling of proteins using biological mass spectrometry (bottom up proteomics) most commonly requires trypsin. Trypsin is advantageous in that it produces peptides of optimal charge and size. However, for applications in which the proteins under investigation are part of a complex mixture or not isolated at high levels (i.e. low ng from an immunoprecipitation), sequence coverage is rarely complete. In addition, we have found that in several cases, like phosphorylation, acetylation, and methylation, alternative proteases are required to prepare peptides suitable for MS detection. This poster will provide specific examples which demonstrate this observation. For example, the application of a combined Trypsin/ Lys-C mixture reduces the number of missed cleavages by more than 3-fold producing samples with lower CV's (for biological replicates). The mixture is also well-suited for the complete proteolysis of hydrophobic, compact proteins. The addition of chymotrypsin and elastase has been found to be useful for identifying phosphorylation sites on proteins, especially on sequences where the site of phosphorylation inhibits trypsin (i.e. proximal to K or R). Many epigenetic applications have focused on histone modifications, like lysine acetylation and arginine methylation. Alternative proteases like Asp-N, Glu-C, and chymotrypsin have been especially useful given the fact that the modified K and R residues are resistant to c-terminal cleavage by trypsin. Finally, in the case of serum profiling, the addition of the endoglycosidase, PNGase F has been found to improve sequence coverage due to the removal of N-linked glycans.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10.

PubMed

Jefferson, Tamara; Auf dem Keller, Ulrich; Bellac, Caroline; Metz, Verena V; Broder, Claudia; Hedrich, Jana; Ohler, Anke; Maier, Wladislaw; Magdolen, Viktor; Sterchi, Erwin; Bond, Judith S; Jayakumar, Arumugam; Traupe, Heiko; Chalaris, Athena; Rose-John, Stefan; Pietrzik, Claus U; Postina, Rolf; Overall, Christopher M; Becker-Pauly, Christoph

2013-01-01

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

PubMed Central

Naito, Yuki; Hino, Kimihiro; Bono, Hidemasa; Ui-Tei, Kumiko

2015-01-01

Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25414360

Rational design of micro-RNA-like bifunctional siRNAs targeting HIV and the HIV coreceptor CCR5.

PubMed

Ehsani, Ali; Saetrom, Pål; Zhang, Jane; Alluin, Jessica; Li, Haitang; Snøve, Ola; Aagaard, Lars; Rossi, John J

2010-04-01

Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3' untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

Photosensitization of DNA damage by a new cationic pyropheophorbide derivative: sequence-specific formation of a frank scission.

PubMed

Kanony, Claire; Fabiano-Tixier, Anne-Sylvie; Ravanat, Jean-Luc; Vicendo, Patricia; Paillous, Nicole

2003-06-01

Pyropheophorbides are red-absorbing porphyrin-like photosensitizers that may interact with DNA either by intercalation or by external binding with self-stacking according to the value of the nucleotide to chromophore molar ratio (N/C). This article reports on the nature and sequence selectivity of the DNA damage photoinduced by a water-soluble chlorhydrate of aminopyropheophorbide. First, this pyropheophorbide is shown to induce on irradiation the cleavage of phiX174 DNA by both Type-I and -II mechanisms, suggested by scavengers and D2O effects. These conclusions are then improved by sequencing experiments performed on a 20-mer oligodeoxynucleotide (ODN) irradiated at wavelengths >345 nm in the presence of the dye, N/C varying from 2.5 to 0.5. Oxidation of all guanine residues to the same extent is observed after piperidine treatment on both single- and double-stranded ODN. Moreover, unexpectedly, a remarkable sequence-selective cleavage occurring at a 5'-CG-3' site is detected before alkali treatment. This frank break is clearly predominant for a low nucleotide to chromophore molar ratio, corresponding to a self-stacking of the dye along the DNA helix. The electrophoretic properties of the band suggest that this lesion results from a sugar oxidation, which leads via a base release to a ribonolactone residue. The proposal is supported by high-performance liquid chromatography-matrix-assisted laser desorption-ionization mass spectrometry experiments that also reveal other sequence-selective frank scissions of lower intensity at 5'-GC-3' or other 5'-CG-3' sites. This sequence selectivity is discussed with regard to the binding selectivity of cationic porphyrins.

Probing the structural dynamics of the CRISPR-Cas9 RNA-guided DNA-cleavage system by coarse-grained modeling.

PubMed

Zheng, Wenjun

2017-02-01

In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Sequence variation in the M2 ion channel protein influences its intracellular localization during avian influenza replication

USDA-ARS?s Scientific Manuscript database

Avian influenza virus (AIV) is a constant threat to poultry worldwide due to its ability to mutate from a low pathogenic (LP) form into a highly pathogenic (HP) one. It is known that the incorporation of a polybasic cleavage site (PBCS) on the hemagglutinin (HA) gene is a major indicator of pathogen...

Retroviral proteases and their roles in virion maturation.

PubMed

Konvalinka, Jan; Kräusslich, Hans-Georg; Müller, Barbara

2015-05-01

Proteolytic processing of viral polyproteins is essential for retrovirus infectivity. Retroviral proteases (PR) become activated during or after assembly of the immature, non-infectious virion. They cleave viral polyproteins at specific sites, inducing major structural rearrangements termed maturation. Maturation converts retroviral enzymes into their functional form, transforms the immature shell into a metastable state primed for early replication events, and enhances viral entry competence. Not only cleavage at all PR recognition sites, but also an ordered sequence of cleavages is crucial. Proteolysis is tightly regulated, but the triggering mechanisms and kinetics and pathway of morphological transitions remain enigmatic. Here, we outline PR structures and substrate specificities focusing on HIV PR as a therapeutic target. We discuss design and clinical success of HIV PR inhibitors, as well as resistance development towards these drugs. Finally, we summarize data elucidating the role of proteolysis in maturation and highlight unsolved questions regarding retroviral maturation. Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

PubMed

Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

2016-03-18

The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

Enhancing Interleukin-6 and Interleukin-11 receptor cleavage.

PubMed

Lokau, Juliane; Wandel, Marieke; Garbers, Christoph

2017-04-01

Proteolytic cleavage of the membrane-bound Interleukin-6 receptor (IL-6R) by the metalloprotease ADAM17 releases an agonistic soluble form of the IL-6R (sIL-6R), which is responsible for the pro-inflammatory trans-signaling branch of the cytokine's activities. This proteolytic step, which is also called ectodomain shedding, is critically regulated by the cleavage site within the IL-6R stalk, because mutations or small deletions within this region are known to render the IL-6R irresponsive towards proteolysis. In the present study, we employed cleavage site profiling data of ADAM17 to generate an IL-6R with increased cleavage susceptibility. Using site-directed mutagenesis, we showed that the non-prime sites P3 and P2 and the prime site P1' were critical for this increase in proteolysis, whereas other positions within the cleavage site were of minor importance. Insertion of this optimized cleavage site into the stalk of the Interleukin-11 receptor (IL-11R) was not sufficient to enable ADAM17-mediated proteolysis, but transfer of different parts of the IL-6R stalk enabled shedding by ADAM17. These findings shed light on the cleavage site specificities of ADAM17 using a native substrate and reveal further differences in the proteolysis of IL-6R and IL-11R. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

PubMed Central

Henderson, L E; Sowder, R; Copeland, T D; Smythers, G; Oroszlan, S

1984-01-01

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus. Images PMID:6333515

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

PubMed

Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

2017-04-06

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

DNA sequence and characterization of GcvA, a LysR family regulatory protein for the Escherichia coli glycine cleavage enzyme system.

PubMed Central

Wilson, R L; Stauffer, G V

1994-01-01

The gene encoding GcvA, the trans-acting regulatory protein for the Escherichia coli glycine cleavage enzyme system, has been sequenced. The gcvA locus contains an open reading frame of 930 nucleotides that could encode a protein with a molecular mass of 34.4 kDa, consistent with the results of minicell analysis indicating that GcvA is a polypeptide of approximately 33 kDa. The deduced amino acid sequence of GcvA revealed that this protein shares similarity with the LysR family of activator proteins. The transcription start site was found to be 72 bp upstream of the presumed translation start site. A chromosomal deletion of gcvA resulted in the inability of cells to activate the expression of a gcvT-lacZ gene fusion when grown in the presence of glycine and an inability to repress gcvT-lacZ expression when grown in the presence of inosine. The regulation of gcvA was examined by constructing a gcvA-lacZ gene fusion in which beta-galactosidase synthesis is under the control of the gcvA regulatory region. Although gcvA expression appears to be autogenously regulated over a two- to threefold range, it is neither induced by glycine nor repressed by inosine. Images PMID:8188587

Amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wheeler, E.F.; Roussel, M.F.; Hampe, A.

1986-08-01

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180/sup gag-fms/ encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180/sup gag-fms/) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence ofmore » the resulting v-fms-coded glycoprotein, gp120/sup v-fms/, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. The authors conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180/gag-fms/ is mediated by signal peptidase and that the amino termini of gp140/sup v-fms/ and the c-fms gene product are identical.« less

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

PubMed Central

Piao, Yi; Kimura, Ayano; Urano, Satomi; Saito, Yuhki; Taru, Hidenori; Yamamoto, Tohru; Hata, Saori; Suzuki, Toshiharu

2013-01-01

Background Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer's β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs. Methodology Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position. Conclusions The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction. PMID:23658629

Cleavage/alteration of interleukin-8 by matrix metalloproteinase-9 in the female lower genital tract.

PubMed

Zariffard, M Reza; Anastos, Kathryn; French, Audrey L; Munyazesa, Elisaphane; Cohen, Mardge; Landay, Alan L; Spear, Gregory T

2015-01-01

Interleukin-8 (IL-8, CXCL8) plays important roles in immune responses at mucosal sites including in the lower genital tract. Since several types of bacteria produce proteases that cleave IL-8 and many types of bacteria can be present in lower genital tract microbiota, we assessed genital fluids for IL-8 cleavage/alteration. Genital fluids collected by lavage from 200 women (23 HIV-seronegative and 177 HIV-seropositive) were tested for IL-8 cleavage/alteration by ELISA. IL-8 cleaving/altering activity was observed in fluids from both HIV-positive (28%) and HIV-negative women (35%). There was no clear relationship between the activity and the types of bacteria present in the lower genital tract as determined by high-throughput sequencing of the 16S rRNA gene. Protease inhibitors specific for matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs. These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract.

A machine learning approach for predicting CRISPR-Cas9 cleavage efficiencies and patterns underlying its mechanism of action.

PubMed

Abadi, Shiran; Yan, Winston X; Amar, David; Mayrose, Itay

2017-10-01

The adaptation of the CRISPR-Cas9 system as a genome editing technique has generated much excitement in recent years owing to its ability to manipulate targeted genes and genomic regions that are complementary to a programmed single guide RNA (sgRNA). However, the efficacy of a specific sgRNA is not uniquely defined by exact sequence homology to the target site, thus unintended off-targets might additionally be cleaved. Current methods for sgRNA design are mainly concerned with predicting off-targets for a given sgRNA using basic sequence features and employ elementary rules for ranking possible sgRNAs. Here, we introduce CRISTA (CRISPR Target Assessment), a novel algorithm within the machine learning framework that determines the propensity of a genomic site to be cleaved by a given sgRNA. We show that the predictions made with CRISTA are more accurate than other available methodologies. We further demonstrate that the occurrence of bulges is not a rare phenomenon and should be accounted for in the prediction process. Beyond predicting cleavage efficiencies, the learning process provides inferences regarding patterns that underlie the mechanism of action of the CRISPR-Cas9 system. We discover that attributes that describe the spatial structure and rigidity of the entire genomic site as well as those surrounding the PAM region are a major component of the prediction capabilities.

Effect of NaeI-L43K mutation on protein dynamics and DNA conformation: Insights from molecular dynamics simulations.

PubMed

Ramachandrakurup, Sreelakshmi; Ramakrishnan, Vigneshwar

2017-09-01

Protein-DNA interactions are an important class of biomolecular interactions inside the cell. Delineating the mechanisms of protein-DNA interactions and more specifically, how proteins search and bind to their specific cognate sequences has been the quest of many in the scientific community. Restriction enzymes have served as useful model systems to this end. In this work, we have investigated using molecular dynamics simulations the effect of L43K mutation on NaeI, a type IIE restriction enzyme. NaeI has two domains, the Topo and the Endo domains, each binding to identical strands of DNA sequences (GCCGGC) 2 . The binding of the DNA to the Topo domain is thought to enhance the binding and cleavage of DNA at the Endo domain. Interestingly, it has been found that the mutation of an amino acid that is distantly-located from the DNA cleavage site (L43K) converts the restriction endonuclease to a topoisomerase. Our investigations reveal that the L43K mutation not only induces local structural changes (as evidenced by changes in hydrogen bond propensities and differences in the percentage of secondary structure assignments of the residues in the ligase-like domain) but also alters the overall protein dynamics and DNA conformation which probably leads to the loss of specific cleavage of the recognition site. In a larger context, our study underscores the importance of considering the role of distantly-located amino acids in understanding protein-DNA interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantification of DNA cleavage specificity in Hi-C experiments.

PubMed

Meluzzi, Dario; Arya, Gaurav

2016-01-08

Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site

PubMed Central

Diederich, Sandra; Berhane, Yohannes; Embury-Hyatt, Carissa; Hisanaga, Tamiko; Handel, Katherine; Cottam-Birt, Colleen; Ranadheera, Charlene; Kobasa, Darwyn

2015-01-01

ABSTRACT Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA0 cleavage site. Furthermore, it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance. PMID:26246579

Signal-3L: A 3-layer approach for predicting signal peptides.

PubMed

Shen, Hong-Bin; Chou, Kuo-Chen

2007-11-16

Functioning as an "address tag" that directs nascent proteins to their proper cellular and extracellular locations, signal peptides have become a crucial tool in finding new drugs or reprogramming cells for gene therapy. To effectively and timely use such a tool, however, the first important thing is to develop an automated method for rapidly and accurately identifying the signal peptide for a given nascent protein. With the avalanche of new protein sequences generated in the post-genomic era, the challenge has become even more urgent and critical. In this paper, we have developed a novel method for predicting signal peptide sequences and their cleavage sites in human, plant, animal, eukaryotic, Gram-positive, and Gram-negative protein sequences, respectively. The new predictor is called Signal-3L that consists of three prediction engines working, respectively, for the following three progressively deepening layers: (1) identifying a query protein as secretory or non-secretory by an ensemble classifier formed by fusing many individual OET-KNN (optimized evidence-theoretic K nearest neighbor) classifiers operated in various dimensions of PseAA (pseudo amino acid) composition spaces; (2) selecting a set of candidates for the possible signal peptide cleavage sites of a query secretory protein by a subsite-coupled discrimination algorithm; (3) determining the final cleavage site by fusing the global sequence alignment outcome for each of the aforementioned candidates through a voting system. Signal-3L is featured by high success prediction rates with short computational time, and hence is particularly useful for the analysis of large-scale datasets. Signal-3L is freely available as a web-server at http://chou.med.harvard.edu/bioinf/Signal-3L/ or http://202.120.37.186/bioinf/Signal-3L, where, to further support the demand of the related areas, the signal peptides identified by Signal-3L for all the protein entries in Swiss-Prot databank that do not have signal peptide annotations or are annotated with uncertain terms but are classified by Signal-3L as secretory proteins are provided in a downloadable file. The large-scale file is prepared with Microsoft Excel and named "Tab-Signal-3L.xls", and will be updated once a year to include new protein entries and reflect the continuous development of Signal-3L.

Global identification of target recognition and cleavage by the Microprocessor in human ES cells

PubMed Central

Seong, Youngmo; Lim, Do-Hwan; Kim, Augustine; Seo, Jae Hong; Lee, Young Sik; Song, Hoseok; Kwon, Young-Soo

2014-01-01

The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein–RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3′ overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. PMID:25326327

Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures1[w

PubMed Central

Loke, Johnny C.; Stahlberg, Eric A.; Strenski, David G.; Haas, Brian J.; Wood, Paul Chris; Li, Qingshun Quinn

2005-01-01

Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3′-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3′-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a single-nucleotide profile scan on the 3′-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which led to a prediction of the formation of secondary structures. Using an RNA secondary structure prediction program, mFold, we identified three main types of secondary structures on the sequences analyzed. Surprisingly, these observed secondary structures were all interrupted in previously constructed mutations in these regions. These results will enable us to revise the current model of plant poly(A) signals and to develop tools to predict 3′-ends for gene annotation. PMID:15965016

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold.

PubMed Central

Rosa, J. C.; De Oliveira, P. S.; Garratt, R.; Beltramini, L.; Resing, K.; Roque-Barreira, M. C.; Greene, L. J.

1999-01-01

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature. PMID:10210179

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element

PubMed Central

2011-01-01

Background The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. Results By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. Conclusions Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host. PMID:21554716

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

PubMed

George, Maja; Schwecke, Torsten; Beimforde, Nadine; Hohn, Oliver; Chudak, Claudia; Zimmermann, Anja; Kurth, Reinhard; Naumann, Dieter; Bannert, Norbert

2011-05-09

The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host.

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease

PubMed Central

Robinson, Clifford R.; Sligar, Stephen G.

1998-01-01

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein–DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein–DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility. PMID:9482860

Coupled transcription and processing of mouse ribosomal RNA in a cell-free system.

PubMed Central

Mishima, Y; Mitsuma, T; Ogata, K

1985-01-01

An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3004977

RNA-dependent RNA targeting by CRISPR-Cas9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

RNA-dependent RNA targeting by CRISPR-Cas9

DOE PAGES

Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine; ...

2018-01-05

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

RNA-dependent RNA targeting by CRISPR-Cas9

PubMed Central

Strutt, Steven C; Torrez, Rachel M; Kaya, Emine; Negrete, Oscar A

2018-01-01

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications. PMID:29303478

DNA Breaks and End Resection Measured Genome-wide by End Sequencing.

PubMed

Canela, Andres; Sridharan, Sriram; Sciascia, Nicholas; Tubbs, Anthony; Meltzer, Paul; Sleckman, Barry P; Nussenzweig, André

2016-09-01

DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice. Published by Elsevier Inc.

Sequence variation between 462 human individuals fine-tunes functional sites of RNA processing

NASA Astrophysics Data System (ADS)

Ferreira, Pedro G.; Oti, Martin; Barann, Matthias; Wieland, Thomas; Ezquina, Suzana; Friedländer, Marc R.; Rivas, Manuel A.; Esteve-Codina, Anna; Estivill, Xavier; Guigó, Roderic; Dermitzakis, Emmanouil; Antonarakis, Stylianos; Meitinger, Thomas; Strom, Tim M.; Palotie, Aarno; François Deleuze, Jean; Sudbrak, Ralf; Lerach, Hans; Gut, Ivo; Syvänen, Ann-Christine; Gyllensten, Ulf; Schreiber, Stefan; Rosenstiel, Philip; Brunner, Han; Veltman, Joris; Hoen, Peter A. C. T.; Jan van Ommen, Gert; Carracedo, Angel; Brazma, Alvis; Flicek, Paul; Cambon-Thomsen, Anne; Mangion, Jonathan; Bentley, David; Hamosh, Ada; Rosenstiel, Philip; Strom, Tim M.; Lappalainen, Tuuli; Guigó, Roderic; Sammeth, Michael

2016-09-01

Recent advances in the cost-efficiency of sequencing technologies enabled the combined DNA- and RNA-sequencing of human individuals at the population-scale, making genome-wide investigations of the inter-individual genetic impact on gene expression viable. Employing mRNA-sequencing data from the Geuvadis Project and genome sequencing data from the 1000 Genomes Project we show that the computational analysis of DNA sequences around splice sites and poly-A signals is able to explain several observations in the phenotype data. In contrast to widespread assessments of statistically significant associations between DNA polymorphisms and quantitative traits, we developed a computational tool to pinpoint the molecular mechanisms by which genetic markers drive variation in RNA-processing, cataloguing and classifying alleles that change the affinity of core RNA elements to their recognizing factors. The in silico models we employ further suggest RNA editing can moonlight as a splicing-modulator, albeit less frequently than genomic sequence diversity. Beyond existing annotations, we demonstrate that the ultra-high resolution of RNA-Seq combined from 462 individuals also provides evidence for thousands of bona fide novel elements of RNA processing—alternative splice sites, introns, and cleavage sites—which are often rare and lowly expressed but in other characteristics similar to their annotated counterparts.

Ab initio DNA synthesis by Bst polymerase in the presence of nicking endonucleases Nt.AlwI, Nb.BbvCI, and Nb.BsmI.

PubMed

Antipova, Valeriya N; Zheleznaya, Lyudmila A; Zyrina, Nadezhda V

2014-08-01

In the absence of added DNA, thermophilic DNA polymerases synthesize double-stranded DNA from free dNTPs, which consist of numerous repetitive units (ab initio DNA synthesis). The addition of thermophilic restriction endonuclease (REase), or nicking endonuclease (NEase), effectively stimulates ab initio DNA synthesis and determines the nucleotide sequence of reaction products. We have found that NEases Nt.AlwI, Nb.BbvCI, and Nb.BsmI with non-palindromic recognition sites stimulate the synthesis of sequences organized mainly as palindromes. Moreover, the nucleotide sequence of the palindromes appeared to be dependent on NEase recognition/cleavage modes. Thus, the heterodimeric Nb.BbvCI stimulated the synthesis of palindromes composed of two recognition sites of this NEase, which were separated by AT-reach sequences or (A)n (T)m spacers. Palindromic DNA sequences obtained in the ab initio DNA synthesis with the monomeric NEases Nb.BsmI and Nt.AlwI contained, along with the sites of these NEases, randomly synthesized sequences consisted of blocks of short repeats. These findings could help investigation of the potential abilities of highly productive ab initio DNA synthesis for the creation of DNA molecules with desirable sequence. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Antibodies to H2a and H2b histones from the sera of HIV-infected patients catalyze site-specific degradation of these histones.

PubMed

Baranova, Svetlana V; Dmitrienok, Pavel S; Ivanisenko, Nikita V; Buneva, Valentina N; Nevinsky, Georgy A

2017-06-01

Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical enzymes were isolated from the sera of HIV-infected patients by chromatography on several affinity sorbents including anti-histone Sepharose. In contrast to canonical proteases (trypsin, chymotrypsin, proteinase K), IgGs from HIV-infected patients specifically hydrolyzed only histones but not many other tested globular proteins. Using MALDI mass spectrometry the sites of H2a and H2b histone cleavage by anti-histone IgGs were determined for the first time. One cluster of H2a hydrolysis contains two major (↕) and four moderate (↓) cleavage sites: 31-H↓R↓L↓L↓R↕K G↕N-38. One major and two moderate sites of cleavage were revealed in the second cluster: 14-A↕KSRS↓SRA↓G-22. The third cluster corresponding to the H2a C-terminal part contains only five minor (†) sites of cleavage: 82-H†LQLAIRNDEELN†KLLG†RV†T†I-102. It was shown that two major and four moderate sites of cleavage were present in the main cluster of H2b hydrolysis: 46-K↕QvhpD↓TgiS↓SkA↓M↕GiM↓N-63. Two moderate sites of cleavage correspond to a relatively short 6-mer cluster: 12-K↓GskK↓A-17. The third relatively long 9-mer cluster contains one major and two minor sites of H2b cleavage: 80-L↕AHYN†KRS†T-88. In the nucleosome core particle, most of the major and moderate cleavage sites are located at the H2a/H2b interaction interface. Minor cleavage sites of H2a are involved in binding with H3 in the nucleosome core. Two moderate cleavage sites of H2b and one major cleavage site of H2a are located in the disordered N-terminal region interacting with DNA. According to the crystal structure of the nucleosome core particle, all identified cleavage sites are expected to affect H2a and H2b folding, nucleosome assembly, and binding of H2a and H2b with DNA. The existence of H2a and H2b hydrolyzing abzymes may be very important for the further understanding of unknown possibilities of immune systems and biological functions of antibodies.

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

PubMed Central

Campbell, T B; Cech, T R

1995-01-01

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library. PMID:7489519

The anti-tumor drug bleomycin preferentially cleaves at the transcription start sites of actively transcribed genes in human cells.

PubMed

Murray, Vincent; Chen, Jon K; Galea, Anne M

2014-04-01

The genome-wide pattern of DNA cleavage at transcription start sites (TSSs) for the anti-tumor drug bleomycin was examined in human HeLa cells using next-generation DNA sequencing. It was found that actively transcribed genes were preferentially cleaved compared with non-transcribed genes. The 143,600 identified human TSSs were split into non-transcribed genes (82,596) and transcribed genes (61,004) for HeLa cells. These transcribed genes were further split into quintiles of 12,201 genes comprising the top 20, 20-40, 40-60, 60-80, and 80-100 % of expressed genes. The bleomycin cleavage pattern at highly transcribed gene TSSs was greatly enhanced compared with purified DNA and non-transcribed gene TSSs. The top 20 and 20-40 % quintiles had a very similar enhanced cleavage pattern, the 40-60 % quintile was intermediate, while the 60-80 and 80-100 % quintiles were close to the non-transcribed and purified DNA profiles. The pattern of bleomycin enhanced cleavage had peaks that were approximately 200 bp apart, and this indicated that bleomycin was identifying the presence of phased nucleosomes at TSSs. Hence bleomycin can be utilized to detect chromatin structures that are present at actively transcribed genes. In this study, for the first time, the pattern of DNA damage by a clinically utilized cancer chemotherapeutic agent was performed on a human genome-wide scale at the nucleotide level.

Creation of a type IIS restriction endonuclease with a long recognition sequence

PubMed Central

Lippow, Shaun M.; Aha, Patti M.; Parker, Matthew H.; Blake, William J.; Baynes, Brian M.; Lipovšek, Daša

2009-01-01

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases. PMID:19304757

Amino acid sequence of tyrosinase from Neurospora crassa.

PubMed Central

Lerch, K

1978-01-01

The amino-acid sequence of tyrosinase from Neurospora crassa (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) is reported. This copper-containing oxidase consists of a single polypeptide chain of 407 amino acids. The primary structure was determined by automated and manual sequence analysis on fragments produced by cleavage with cyanogen bromide and on peptides obtained by digestion with trypsin, pepsin, thermolysin, or chymotrypsin. The amino terminus of the protein is acetylated and the single cysteinyl residue 96 is covalently linked via a thioether bridge to histidyl residue 94. The formation and the possible role of this unusual structure in Neurospora tyrosinase is discussed. Dye-sensitized photooxidation of apotyrosinase and active-site-directed inactivation of the native enzyme indicate the possible involvement of histidyl residues 188, 192, 289, and 305 or 306 as ligands to the active-site copper as well as in the catalytic mechanism of this monooxygenase. PMID:151279

Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: determinants and constraints.

PubMed

Lai, Zon W; Hanchapola, Iresha; Steer, David L; Smith, A Ian

2011-06-14

ADAM17, also known as tumor necrosis factor α-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) → Glu(708) mutation and the Arg(708)Arg(710) → Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.

Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

PubMed

Gimble, F S; Thorner, J

1993-10-15

The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

Different Requirements for Proteolytic Processing of Bone Morphogenetic Protein 5/6/7/8 Ligands in Drosophila melanogaster*

PubMed Central

Fritsch, Cornelia; Sawala, Annick; Harris, Robin; Maartens, Aidan; Sutcliffe, Catherine; Ashe, Hilary L.; Ray, Robert P.

2012-01-01

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species. PMID:22199351

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.

PubMed

Gasparian, Marine E; Bobik, Tatyana V; Kim, Yana V; Ponomarenko, Natalia A; Dolgikh, Dmitry A; Gabibov, Alexander G; Kirpichnikov, Mikhail P

2013-11-01

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 μM and 20 μM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Pathotypic characterization of Newcastle disease virus isolated from vaccinated chicken in West Java, Indonesia.

PubMed

Putri, Dwi Desmiyeni; Handharyani, Ekowati; Soejoedono, Retno Damajanti; Setiyono, Agus; Mayasari, Ni Luh Putu Ika; Poetri, Okti Nadia

2017-04-01

This research was conducted to differentiate and characterize eight Newcastle disease virus (NDV) isolates collected from vaccinated chicken at commercial flocks in West Java, Indonesia, in 2011, 2014 and 2015 by pathotype specific primers. A total of eight NDV isolates collected from clinical outbreaks among commercial vaccinated flocks in West Java, Indonesia, in 2011, 2014, and 2015 were used in this study. Reverse transcription-polymerase chain reaction was used to detect and differentiate virulence of NDV strains, using three sets of primers targeting their M and F gene. First primers were universal primers to detect NDV targeting matrix (M) gene. Other two sets of primers were specific for the fusion (F) gene cleavage site sequence of virulent and avirulent NDV strains. Our results showed that three isolates belong to NDV virulent strains, and other five isolates belong to NDV avirulent strains. The nucleotide sequence of the F protein cleavage site showed 112 K/R-R-Q/R-K-R/G-F 117 on NDV virulent strains and 112 G-K/R-Q-G-R-L 117 on NDV avirulent strain. Result from the current study suggested that NDV virulent strain were circulating among vaccinated chickens in West Java, Indonesia; this might possess a risk of causing ND outbreaks and causing economic losses within the poultry industry.

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

PubMed Central

Chand, Mahesh Kumar; Nirwan, Neha; Diffin, Fiona M.; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D.; Saikrishnan, Kayarat

2015-01-01

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission. PMID:26389736

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

PubMed

Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of amore » large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.« less

Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region.

PubMed Central

Gaynor, R; Soultanakis, E; Kuwabara, M; Garcia, J; Sigman, D S

1989-01-01

The transactivator protein, tat, encoded by the human immunodeficiency virus is a key regulator of viral transcription. Activation by the tat protein requires sequences downstream of the transcription initiation site called the transactivating region (TAR). RNA derived from the TAR is capable of forming a stable stem-loop structure and the maintenance of both the stem structure and the loop sequences located between +19 and +44 is required for complete in vivo activation by tat. Gel retardation assays with RNA from both wild-type and mutant TAR constructs generated in vitro with SP6 polymerase indicated specific binding of HeLa nuclear proteins to the TAR. To characterize this RNA-protein interaction, a method of chemical "imprinting" has been developed using photoactivated uranyl acetate as the nucleolytic agent. This reagent nicks RNA under physiological conditions at all four nucleotides in a reaction that is independent of sequence and secondary structure. Specific interaction of cellular proteins with TAR RNA could be detected by enhanced cleavages or imprints surrounding the loop region. Mutations that either disrupted stem base-pairing or extensively changed the primary sequence resulted in alterations in the cleavage pattern of the TAR RNA. Structural features of the TAR RNA stem-loop essential for tat activation are also required for specific binding of the HeLa cell nuclear protein. Images PMID:2544877

High-throughput detection of RNA processing in bacteria.

PubMed

Gill, Erin E; Chan, Luisa S; Winsor, Geoffrey L; Dobson, Neil; Lo, Raymond; Ho Sui, Shannan J; Dhillon, Bhavjinder K; Taylor, Patrick K; Shrestha, Raunak; Spencer, Cory; Hancock, Robert E W; Unrau, Peter J; Brinkman, Fiona S L

2018-03-27

Understanding the RNA processing of an organism's transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5'-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions. The pRNA-Seq library revealed known tRNA, rRNA and transfer-messenger RNA (tmRNA) processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5' ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and were centered on single palindromic TAT(A/T)ATA motifs (likely - 10 promoter elements), suggesting that, consistent with previous in vitro experimentation, these sites can initiate transcription bi-directionally and may thus provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions. This study uses pRNA-Seq, a method that provides a genome-wide survey of RNA processing, to study the bacterium Pseudomonas aeruginosa and discover extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation. NOTE: All sequence data has been submitted to the NCBI sequence read archive. Accession numbers are as follows: [NCBI sequence read archive: SRX156386, SRX157659, SRX157660, SRX157661, SRX157683 and SRX158075]. The sequence data is viewable using Jbrowse on www.pseudomonas.com .

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

PubMed Central

Martin, Annette; Bénichou, Danièle; Chao, Shih-Fong; Cohen, Lisette M.; Lemon, Stanley M.

1999-01-01

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase. PMID:10400711

A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometry.

PubMed

Diago-Navarro, Elizabeth; Kamphuis, Monique B; Boelens, Rolf; Barendregt, Arjan; Heck, Albert J; van den Heuvel, Robert H; Díaz-Orejas, Ramón

2009-09-01

Kid, the toxin of the parD (kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5' of A in the core sequence 5'-UA(A/C)-3'. A model of the Kid toxin interacting with the uncleavable mimetic 5'-AdUACA-3' is available. To evaluate this model, a significant collection of mutants in some of the key residues proposed to be involved in RNA binding (T46, A55, T69 and R85) or RNA cleavage (R73, D75 and H17) were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates, 5'-AUACA-3', and its uncleavable mimetic 5'-AdUACA-3', used to establish the model and structure of the Kid-RNA complex, were used in both the RNA cleavage and binding assays. A second RNA substrate, 5'-UUACU-3' efficiently cleaved by Kid both in vivo and in vitro, was also used in the cleavage assays. Compared with the wild-type protein, mutations in the residues of the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5'-UUACU-3' substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid-RNA complex.

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates

PubMed Central

Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

2005-01-01

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain. PMID:16262906

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

PubMed

Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

2005-11-01

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

The Effects of Trivalent Lanthanide Cationization on the Electron Transfer Dissociation of Acidic Fibrinopeptide B and its Analogs

NASA Astrophysics Data System (ADS)

Commodore, Juliette J.; Cassady, Carolyn J.

2016-09-01

Electrospray ionization (ESI) on mixtures of acidic fibrinopeptide B and two peptide analogs with trivalent lanthanide salts generates [M + Met + H]4+, [M + Met]3+, and [M + Met -H]2+, where M = peptide and Met = metal (except radioactive promethium). These ions undergo extensive and highly efficient electron transfer dissociation (ETD) to form metallated and non-metallated c- and z-ions. All metal adducted product ions contain at least two acidic sites, which suggest attachment of the lanthanide cation at the side chains of one or more acidic residues. The three peptides undergo similar fragmentation. ETD on [M + Met + H]4+ leads to cleavage at every residue; the presence of both a metal ion and an extra proton is very effective in promoting sequence-informative fragmentation. Backbone dissociation of [M + Met]3+ is also extensive, although cleavage does not always occur between adjacent glutamic acid residues. For [M + Met - H ]2+, a more limited range of product ions form. All lanthanide metal peptide complexes display similar fragmentation except for europium (Eu). ETD on [M + Eu - H]2+ and [M + Eu]3+ yields a limited amount of peptide backbone cleavage; however, [M + Eu + H]4+ dissociates extensively with cleavage at every residue. With the exception of the results for Eu(III), metallated peptide ion formation by ESI, ETD fragmentation efficiencies, and product ion formation are unaffected by the identity of the lanthanide cation. Adduction with trivalent lanthanide metal ions is a promising tool for sequence analysis of acidic peptides by ETD.

Nop9 is a PUF-like protein that prevents premature cleavage to correctly process pre-18S rRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jun; McCann, Kathleen L.; Qiu, Chen

Numerous factors direct eukaryotic ribosome biogenesis, and defects in a single ribosome assembly factor may be lethal or produce tissue-specific human ribosomopathies. Pre-ribosomal RNAs (pre-rRNAs) must be processed stepwise and at the correct subcellular locations to produce the mature rRNAs. Nop9 is a conserved small ribosomal subunit biogenesis factor, essential in yeast. Here we report a 2.1-Å crystal structure of Nop9 and a small-angle X-ray-scattering model of a Nop9:RNA complex that reveals a ‘C’-shaped fold formed from 11 Pumilio repeats. We show that Nop9 recognizes sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease,more » Nob1. We further demonstrate that Nop9 inhibits Nob1 cleavage, the final processing step to produce mature small ribosomal subunit 18S rRNA. Together, our results suggest that Nop9 is critical for timely cleavage of the 20S pre-rRNA. Moreover, the Nop9 structure exemplifies a new class of Pumilio repeat proteins.« less

Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

PubMed Central

Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

2015-01-01

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

Model for RNA binding and the catalytic site of the RNase Kid of the bacterial parD toxin-antitoxin system.

PubMed

Kamphuis, Monique B; Bonvin, Alexandre M J J; Monti, Maria Chiara; Lemonnier, Marc; Muñoz-Gómez, Ana; van den Heuvel, Robert H H; Díaz-Orejas, Ramón; Boelens, Rolf

2006-03-17

The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.

Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

PubMed

Heilmann, L J; Trewitt, P M; Kumaran, A K

1993-01-01

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

Global identification of target recognition and cleavage by the Microprocessor in human ES cells.

PubMed

Seong, Youngmo; Lim, Do-Hwan; Kim, Augustine; Seo, Jae Hong; Lee, Young Sik; Song, Hoseok; Kwon, Young-Soo

2014-11-10

The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3' overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

PubMed Central

Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

2010-01-01

BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

PubMed

Joseph, Thomas T; Osman, Roman

2012-01-01

In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of a large number of internal allosteric pathways.

Convergent Transmission of RNAi Guide-Target Mismatch Information across Argonaute Internal Allosteric Network

PubMed Central

Joseph, Thomas T.; Osman, Roman

2012-01-01

In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand “seed region” have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of a large number of internal allosteric pathways. PMID:23028290

Probing the hammerhead ribozyme structure with ribonucleases.

PubMed Central

Hodgson, R A; Shirley, N J; Symons, R H

1994-01-01

Susceptibility to RNase digestion has been used to probe the conformation of the hammerhead ribozyme structure prepared from chemically synthesised RNAs. Less than about 1.5% of the total sample was digested to obtain a profile of RNase digestion sites. The observed digestion profiles confirmed the predicted base-paired secondary structure for the hammerhead. Digestion profiles of both cis and trans hammerhead structures were nearly identical which indicated that the structural interactions leading to self-cleavage were similar for both systems. Furthermore, the presence or absence of Mg2+ did not affect the RNase digestion profiles, thus indicating that Mg2+ did not modify the hammerhead structure significantly to induce self-cleavage. The base-paired stems I and II in the hammerhead structure were stable whereas stem III, which was susceptible to digestion, appeared to be an unstable region. The single strand domains separating the stems were susceptible to digestion with the exception of sites adjacent to guanosines; GL2.1 in the stem II loop and G12 in the conserved GAAAC sequence, which separates stems II and III. The absence of digestion at GL2.1 in the stem II hairpin loop of the hammerhead complex was maintained in uncomplexed ribozyme and in short oligonucleotides containing only the stem II hairpin region. In contrast, the G12 site became susceptible when the ribozyme was not complexed with its substrate. Overall the results are consistent with the role of Mg2+ in the hammerhead self-cleavage reaction being catalytic and not structural. Images PMID:8202361

Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase.

PubMed Central

Allawi, H T; Dong, F; Ip, H S; Neri, B P; Lyamichev, V I

2001-01-01

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility. PMID:11233988

Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

PubMed

Liu, Xiaochuan; Freitas, Jaime; Zheng, Dinghai; Oliveira, Marta S; Hoque, Mainul; Martins, Torcato; Henriques, Telmo; Tian, Bin; Moreira, Alexandra

2017-12-01

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster , expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis -element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215 C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate. © 2017 Liu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The sRNAome mining revealed existence of unique signature small RNAs derived from 5.8SrRNA from Piper nigrum and other plant lineages.

PubMed

Asha, Srinivasan; Soniya, E V

2017-02-01

Small RNAs derived from ribosomal RNAs (srRNAs) are rarely explored in the high-throughput data of plant systems. Here, we analyzed srRNAs from the deep-sequenced small RNA libraries of Piper nigrum, a unique magnoliid plant. The 5' end of the putative long form of 5.8S rRNA (5.8S L rRNA) was identified as the site for biogenesis of highly abundant srRNAs that are unique among the Piperaceae family of plants. A subsequent comparative analysis of the ninety-seven sRNAomes of diverse plants successfully uncovered the abundant existence and precise cleavage of unique rRF signature small RNAs upstream of a novel 5' consensus sequence of the 5.8S rRNA. The major cleavage process mapped identically among the different tissues of the same plant. The differential expression and cleavage of 5'5.8S srRNAs in Phytophthora capsici infected P. nigrum tissues indicated the critical biological functions of these srRNAs during stress response. The non-canonical short hairpin precursor structure, the association with Argonaute proteins, and the potential targets of 5'5.8S srRNAs reinforced their regulatory role in the RNAi pathway in plants. In addition, this novel lineage specific small RNAs may have tremendous biological potential in the taxonomic profiling of plants.

The sRNAome mining revealed existence of unique signature small RNAs derived from 5.8SrRNA from Piper nigrum and other plant lineages

PubMed Central

Asha, Srinivasan; Soniya, E. V.

2017-01-01

Small RNAs derived from ribosomal RNAs (srRNAs) are rarely explored in the high-throughput data of plant systems. Here, we analyzed srRNAs from the deep-sequenced small RNA libraries of Piper nigrum, a unique magnoliid plant. The 5′ end of the putative long form of 5.8S rRNA (5.8SLrRNA) was identified as the site for biogenesis of highly abundant srRNAs that are unique among the Piperaceae family of plants. A subsequent comparative analysis of the ninety-seven sRNAomes of diverse plants successfully uncovered the abundant existence and precise cleavage of unique rRF signature small RNAs upstream of a novel 5′ consensus sequence of the 5.8S rRNA. The major cleavage process mapped identically among the different tissues of the same plant. The differential expression and cleavage of 5′5.8S srRNAs in Phytophthora capsici infected P. nigrum tissues indicated the critical biological functions of these srRNAs during stress response. The non-canonical short hairpin precursor structure, the association with Argonaute proteins, and the potential targets of 5′5.8S srRNAs reinforced their regulatory role in the RNAi pathway in plants. In addition, this novel lineage specific small RNAs may have tremendous biological potential in the taxonomic profiling of plants. PMID:28145468

Evolution of a mass spectrometry-grade protease with PTM-directed specificity.

PubMed

Tran, Duc T; Cavett, Valerie J; Dang, Vuong Q; Torres, Héctor L; Paegel, Brian M

2016-12-20

Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (k cat /K M = 6.9 × 10 5 M -1 ⋅s -1 ). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y 1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.

Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodi, D. J.; Soares, A. S.; Makowski, L.

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usagemore » patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.« less

The identification of disulfides in ricin D using proteolytic cleavage followed by negative-ion nano-electrospray ionization mass spectrometry of the peptide fragments.

PubMed

Tran, T T Nha; Brinkworth, Craig S; Bowie, John H

2015-01-30

To use negative-ion nano-electrospray ionization mass spectrometry of peptides from the tryptic digest of ricin D, to provide sequence information; in particular, to identify disulfide position and connectivity. Negative-ion fragmentations of peptides from the tryptic digest of ricin D was studied using a Waters QTOF2 mass spectrometer operating in MS and MS(2) modes. Twenty-three peptides were obtained following high-performance liquid chromatography and studied by negative-ion mass spectrometry covering 73% of the amino-acid residues of ricin D. Five disulfide-containing peptides were identified, three intermolecular and two intramolecular disulfide-containing peptides. The [M-H](-) anions of the intermolecular disulfides undergo facile cleavage of the disulfide units to produce fragment peptides. In negative-ion collision-induced dissociation (CID) these source-formed anions undergo backbone cleavages, which provide sequencing information. The two intramolecular disulfides were converted proteolytically into intermolecular disulfides, which were identified as outlined above. The positions of the five disulfide groups in ricin D may be determined by characteristic negative-ion cleavage of the disulfide groups, while sequence information may be determined using the standard negative-ion backbone cleavages of the resulting cleaved peptides. Negative-ion mass spectrometry can also be used to provide partial sequencing information for other peptides (i.e. those not containing Cys) using the standard negative-ion backbone cleavages of these peptides. Copyright © 2014 John Wiley & Sons, Ltd.

The Medicinal Chemistry of Botulinum, Ricin and Anthrax Toxins

DTIC Science & Technology

2005-01-01

sites for two urban population of 5 million would yield 250,000 exposed calcium ions as well as the cleavage site for the furin persons and result in...proteolytic cleavage by furin , or a dosage required, and whether or not concomitant vaccination furin -like protease, at a surface loop within Domain 1...existing mainly as antiparallel l3-sheets [128,129]. Domain I, contains the binding sites for two calcium ions as well as the cleavage site for the furin

An att site-based recombination reporter system for genome engineering and synthetic DNA assembly.

PubMed

Bland, Michael J; Ducos-Galand, Magaly; Val, Marie-Eve; Mazel, Didier

2017-07-14

Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla). The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested. The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP.

PubMed

Sardana, Richa; White, Joshua P; Johnson, Arlen W

2013-06-01

Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

PubMed Central

Ethier, Sylvain; Schmeing, T. Martin; Dostie, Josée; Pelletier, Jerry

2014-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data. PMID:25275497

Intermolecular cleavage by UmuD-like mutagenesis proteins

PubMed Central

McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

1998-01-01

The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

Comparison of Methods of Detection of Exceptional Sequences in Prokaryotic Genomes.

PubMed

Rusinov, I S; Ershova, A S; Karyagina, A S; Spirin, S A; Alexeevski, A V

2018-02-01

Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under- or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subsequences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes.

γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP).

PubMed

Lessard, Christian B; Cottrell, Barbara A; Maruyama, Hiroko; Suresh, Suraj; Golde, Todd E; Koo, Edward H

2015-01-01

The relative increase in Aβ42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid β-protein (Aβ) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aβ peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aβ42/Aβ40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aβ42 generation by altering processivity but not ε-cleavage site utilization.

GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

PubMed Central

Nguyen, Nhu T.; Liebers, Matthew; Topkar, Ved V.; Thapar, Vishal; Wyvekens, Nicolas; Khayter, Cyd; Iafrate, A. John; Le, Long P.; Aryee, Martin J.; Joung, J. Keith

2014-01-01

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called Genome-wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-Seq), relies on capture of double-stranded oligodeoxynucleotides into breaks Application of GUIDE-Seq to thirteen RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or ChIP-Seq. GUIDE-Seq also identified RGN-independent genomic breakpoint ‘hotspots’. Finally, GUIDE-Seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases prior to clinical use. PMID:25513782

StarScan: a web server for scanning small RNA targets from degradome sequencing data.

PubMed

Liu, Shun; Li, Jun-Hao; Wu, Jie; Zhou, Ke-Ren; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

2015-07-01

Endogenous small non-coding RNAs (sRNAs), including microRNAs, PIWI-interacting RNAs and small interfering RNAs, play important gene regulatory roles in animals and plants by pairing to the protein-coding and non-coding transcripts. However, computationally assigning these various sRNAs to their regulatory target genes remains technically challenging. Recently, a high-throughput degradome sequencing method was applied to identify biologically relevant sRNA cleavage sites. In this study, an integrated web-based tool, StarScan (sRNA target Scan), was developed for scanning sRNA targets using degradome sequencing data from 20 species. Given a sRNA sequence from plants or animals, our web server performs an ultrafast and exhaustive search for potential sRNA-target interactions in annotated and unannotated genomic regions. The interactions between small RNAs and target transcripts were further evaluated using a novel tool, alignScore. A novel tool, degradomeBinomTest, was developed to quantify the abundance of degradome fragments located at the 9-11th nucleotide from the sRNA 5' end. This is the first web server for discovering potential sRNA-mediated RNA cleavage events in plants and animals, which affords mechanistic insights into the regulatory roles of sRNAs. The StarScan web server is available at http://mirlab.sysu.edu.cn/starscan/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Purification and characterization of a cysteine protease from corms of freesia, Freesia reflacta.

PubMed

Kaneda, M; Yonezawa, H; Uchikoba, T

1997-09-01

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M(r) was estimated to be about 26,000 by SDS-PAGE. The optimum pH of the enzyme was 6.0-7.0 at 30 degrees C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at P1 positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P2 position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.

RNase P cleaves transient structures in some riboswitches.

PubMed

Altman, Sidney; Wesolowski, Donna; Guerrier-Takada, Cecilia; Li, Yong

2005-08-09

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5' UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5' UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested.

RNase P cleaves transient structures in some riboswitches

PubMed Central

Altman, Sidney; Wesolowski, Donna; Guerrier-Takada, Cecilia; Li, Yong

2005-01-01

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5′ UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5′ UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested. PMID:16061811

Co-circulation of paramyxo- and influenza viruses in pigeons in Egypt.

PubMed

Mansour, Shimaa M G; Mohamed, Fakry F; Eid, Amal A M; Mor, Sunil K; Goyal, Sagar M

2017-08-01

In recent years, avian influenza virus (AIV) and Newcastle disease virus (NDV) have caused large-scale outbreaks in many countries, including Egypt. The culling and vaccination strategies have failed to control both viruses in Egypt. In this study, we investigated the outbreaks of nervous manifestations and deaths in pigeons between 2013 and 2015. The H5N1 subtype of the highly pathogenic avian influenza virus and pigeon paramyxovirus-1, an antigenic variant of NDV, were found to be the cause; AIV and pigeon paramyxovirus-1 were isolated from 61.3% (19/31) and 67.8% (21/31) of tested pigeons, respectively. Co-infection with both viruses was detected in 51.6% of pigeons (16/31). The AIV sequences showed PQGEKRRKKR/GLF motif at the haemagglutinin gene cleavage site, which is typical of the highly pathogenic H5N1 subtype. The phylogenetic tree showed that the highly pathogenic avian influenza belonged to clade 2.2.1.2. The NDV sequences carried one of the three motifs, 112 GKQGRL 117 , 112 KRQKRF 117 or 112 RRQKRF 117 , at the fusion protein cleavage site and were classified as genotypes I, VI and II in NDV-class II, respectively. This indicated that different genotypes of NDV can circulate simultaneously among pigeons. Further analysis revealed the clustering of some sequences in sub-genotypes Ia and VIb.2. To the best of our knowledge, these sub-genotypes have not been previously reported from pigeons in Egypt. Our results should serve as a base for future studies on both viruses in Egypt.

Programmable DNA-Guided Artificial Restriction Enzymes.

PubMed

Enghiad, Behnam; Zhao, Huimin

2017-05-19

Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research. However, they have limited sequence specificity and availability. Here we report a Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by reannealing of the cleaved single stranded DNAs. We used this platform to generate over 18 AREs for DNA fingerprinting and molecular cloning of PCR-amplified or genomic DNAs. These AREs work as efficiently as their naturally occurring counterparts, and some of them even do not have any naturally occurring counterparts, demonstrating easy programmability, generality, versatility, and high efficiency for this new technology.

Display of adenoregulin with a novel Pichia pastoris cell surface display system.

PubMed

Ren, Ren; Jiang, Zhengbing; Liu, Meiyun; Tao, Xinyi; Ma, Yushu; Wei, Dongzhi

2007-02-01

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo1p with its own secretion signal sequence or the alpha-factor secretion signal sequence, a polyhistidine (6xHis) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.

Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

PubMed

Tuan, Pham Anh; Park, Sang Un

2013-01-01

Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress. Copyright © 2012 Elsevier GmbH. All rights reserved.

Engineering Environmentally-Stable Proteases to Specifically Neutralize Protein Toxins

DTIC Science & Technology

2013-10-01

acids. These sites constitute a variable environment, with the effect of mutations largely isolated to effects on interactions with the P4 side chain. 2...desires to cut. We observe, however, sequence-specific cleavage is much more subtle, depending upon how side chain interactions influence not only...first five substrate amino acids on the acyl side of the scissile bond (denoted P1 through P5, numbering from the scissile bond toward the N-terminus

Engineering Environmentally-Stable Proteases to Specifically Neutralize Protein Toxins

DTIC Science & Technology

2012-10-14

effect of mutations largely isolated to effects on interactions with the P4 side chain. 2) Most mutations at some sites (e.g. 126, 128) decrease...to cut. We observe, however, sequence-specific cleavage is much more subtle, depending upon how side chain interactions influence not only ground...five substrate amino acids on the acyl side of the scissile bond (denoted P1 through P5, numbering from the scissile bond toward the N-terminus of the

Caspase-1 and IL-1β Processing in a Teleost Fish

PubMed Central

Reis, Marta I. R.; do Vale, Ana; Pereira, Pedro J. B.; Azevedo, Jorge E.; dos Santos, Nuno M. S.

2012-01-01

Interleukine-1β (IL-1β) is the most studied pro-inflammatory cytokine, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges. In mammals, IL-1β is synthesized as an inactive 31 kDa precursor that is cleaved by caspase-1 generating a 17.5 kDa secreted active mature form. The caspase-1 cleavage site strictly conserved in all mammalian IL-1β sequences is absent in IL-1β sequences reported for non-mammalian vertebrates. Recently, fish caspase-1 orthologues have been identified in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) but very little is known regarding their processing and activity. In this work it is shown that sea bass caspase-1 auto-processing is similar to that of the human enzyme, resulting in active p24/p10 and p20/p10 heterodimers. Moreover, the presence of alternatively spliced variants of caspase-1 in sea bass is reported. The existence of caspase-1 isoforms in fish and in mammals suggests that they have been evolutionarily maintained and therefore are likely to play a regulatory role in the inflammatory response, as shown for other caspases. Finally, it is shown that sea bass and avian IL-1β are specifically cleaved by caspase-1 at different but phylogenetically conserved aspartates, distinct from the cleavage site of mammalian IL-1β. PMID:23226286

Characterization of two pigeon paramyxovirus type 1 isolates in China.

PubMed

Awu, Abie; Shao, Meng-yu; Liu, Meng-meng; Hu, Yan-xin; Qin, Zhuo-ming; Tian, Fu-lin; Zhang, Guo-zhong

2015-01-01

For over three decades, there has been a continuing panzootic caused by a virulent variant avian paramyxovirus type 1 strain, the so-called pigeon paramyxovirus type 1. It is found primarily in racing pigeons, but it has also spread to wild birds and poultry. In this study, two pigeon paramyxovirus type 1 strains, SD12 and BJ13, obtained from diseased pigeons in China, were characterized. Phylogenetic analysis based on complete sequences allowed characterization of both strains as genotype VI, class II. Further phylogenetic analysis of a 374-nucleotide section of the fusion gene showed that SD12 fell into lineage VIbii-d and BJ13 into VIbii-f. The deduced amino acid sequence of the cleavage site of the fusion protein confirmed that both isolates contained the virulent motif (112)K/RRQKR↓F(117) at the cleavage site. Nevertheless, the values of intracerebral pathogenicity indices showed the SD12 isolate to be a velogenic strain and BJ13 isolate to be a mesogenic strain. The SD12 isolate was further investigated via clinical observation, RNA detection, histopathology and viral serology in experimentally infected 3-week-old chickens. It showed a mild pathological phenotype in chickens, with viral replication restricted to a few tissues. The molecular mechanism for the SD12 isolate to have a virulent motif but low levels of virulence for chickens requires further study.

REBASE--a database for DNA restriction and modification: enzymes, genes and genomes.

PubMed

Roberts, Richard J; Vincze, Tamas; Posfai, Janos; Macelis, Dana

2015-01-01

REBASE is a comprehensive and fully curated database of information about the components of restriction-modification (RM) systems. It contains fully referenced information about recognition and cleavage sites for both restriction enzymes and methyltransferases as well as commercial availability, methylation sensitivity, crystal and sequence data. All genomes that are completely sequenced are analyzed for RM system components, and with the advent of PacBio sequencing, the recognition sequences of DNA methyltransferases (MTases) are appearing rapidly. Thus, Type I and Type III systems can now be characterized in terms of recognition specificity merely by DNA sequencing. The contents of REBASE may be browsed from the web http://rebase.neb.com and selected compilations can be downloaded by FTP (ftp.neb.com). Monthly updates are also available via email. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

CRISPR/Cas9 for genome editing: progress, implications and challenges.

PubMed

Zhang, Feng; Wen, Yan; Guo, Xiong

2014-09-15

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific genomic elements, and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9 comprises of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA), which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA repair process leads to desired insertions, deletions or substitutions at target sites. The specificity of CRISPR/Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif locating at downstream of target sequences. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome editing and clinical therapeutic potential of CRISPR/Cas9 in future. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Formation of Pmel17 Amyloid Is Regulated by Juxtamembrane Metalloproteinase Cleavage, and the Resulting C-terminal Fragment Is a Substrate for γ-Secretase*

PubMed Central

Kummer, Markus P.; Maruyama, Hiroko; Huelsmann, Claudia; Baches, Sandra; Weggen, Sascha; Koo, Edward H.

2009-01-01

The formation of insoluble cross β-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MαC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the γ-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas γ-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position. PMID:19047044

A DNAzyme requiring two different metal ions at two distinct sites

PubMed Central

Zhou, Wenhu; Zhang, Yupei; Huang, Po-Jung Jimmy; Ding, Jinsong; Liu, Juewen

2016-01-01

Most previously reported RNA-cleaving DNAzymes require only a single divalent metal ion for catalysis. We recently reported a general trivalent lanthanide-dependent DNAzyme named Ce13d. This work shows that Ce13d requires both Na+ and a trivalent lanthanide (e.g. Ce3+), simultaneously. This discovery is facilitated by the sequence similarity between Ce13d and a recently reported Na+-specific DNAzyme, NaA43. The Ce13d cleavage rate linearly depends on the concentration of both metal ions. Sensitized Tb3+ luminescence and DMS footprinting experiments indicate that the guanines in the enzyme loop are important for Na+-binding. The Na+ dissociation constants of Ce13d measured from the cleavage activity assay, Tb3+ luminescence and DMS footprinting are 24.6, 16.3 and 47 mM, respectively. Mutation studies indicate that the role of Ce3+ might be replaced by G23 in NaA43. Ce3+ functions by stabilizing the transition state phosphorane, thus promoting cleavage. G23 competes favorably with low concentration Ce3+ (below 1 μM). The G23-to-hypoxanthine mutation suggests the N1 position of the guanine as a hydrogen bond donor. Together, Ce13d has two distinct metal binding sites, each fulfilling a different role. DNAzymes can be quite sophisticated in utilizing metal ions for catalysis and molecular recognition, similar to protein metalloenzymes. PMID:26657636

Association of I-FABP gene polymorphism and the risk of coronary heart disease.

PubMed

Yuan, Dong; Yu, Changqing; Zeng, Chunyu

2015-01-01

The study aimed to investigate the association between polymorphism of I-FABP gene and coronary heart disease (CHD). 225 patients with CHD were randomly recruited into the case group, and 196 healthy elderly volunteers were recruited from Medical Examination Center of our hospital as control. General clinical data were collected and plasma TC, TG, LDL-C, HDL-C levels were measured. Besides, polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) technology were used to detect the polymorphism of Hha-I enzyme cleavage sites in I-FABP gene in the study population. Hha-I cleavage sites occurred at codon 54 in exon in the coding sequencing of I-FABP gene in all participants. After cleavage with Hha-I enzyme, the genotypes were identified as wild-type A/A, heterozygous mutant A/T and homozygous mutant T/T. In case group, A/T and T/T genetic carriers had significantly higher levels of TC, TG and LDL-C than A/A carriers (P<0.05). However, in control group, similar differences were not observed (P>0.05). BMI, dietary habits and I-FABP alleles were independent risk factors of CHD. The polymorphism of I-FABP gene existed in the study population. And this genetic variation had influence on lipid metabolism, which was associated with the risk of developing CHD. I-FABP gene polymorphism may contribute to the increased genetic susceptibility to CHD.

Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery.

PubMed

Donovan, Jesse; Rath, Sneha; Kolet-Mandrikov, David; Korennykh, Alexei

2017-11-01

Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest. © 2017 Donovan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

UVA irradiation of BrU-substituted DNA in the presence of Hoechst 33258.

PubMed

Saha, Abhijit; Kizaki, Seiichiro; Han, Ji Hoon; Yu, Zutao; Sugiyama, Hiroshi

2018-01-01

Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil ( Br U)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to Br U caused DNA cleavage preferentially at self-complementary 5'-AA Br U Br U-3' sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

PubMed

Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

2014-08-01

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis

PubMed Central

Mancini, Marie; Machamer, Carolyn E.; Roy, Sophie; Nicholson, Donald W.; Thornberry, Nancy A.; Casciola-Rosen, Livia A.; Rosen, Antony

2000-01-01

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors. PMID:10791974

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

PubMed Central

Kramer, K M; Brock, J A; Bloom, K; Moore, J K; Haber, J E

1994-01-01

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells. PMID:8289808

A carboxy-terminal fragment of protein mu 1/mu 1C is present in infectious subvirion particles of mammalian reoviruses and is proposed to have a role in penetration.

PubMed Central

Nibert, M L; Fields, B N

1992-01-01

Penetration of a cell membrane as an early event in infection of cells by mammalian reoviruses appears to require a particular type of viral particle, the infectious subvirion particle (ISVP), which is generated from an intact virion by proteolytic cleavage of the outer capsid proteins sigma 3 and mu 1/mu 1C. Characterizations of the structural components and properties of ISVPs are thus relevant to attempts to understand the mechanism of penetration by reoviruses. In this study, a novel, approximately 13-kDa carboxy-terminal fragment (given the name phi) was found to be generated from protein mu 1/mu 1C during in vitro treatments of virions with trypsin or chymotrypsin to yield ISVPs. With trypsin treatment, both the carboxy-terminal fragment phi and the amino-terminal fragment mu 1 delta/delta were shown to be generated and to remain attached to ISVPs in stoichiometric quantities. Sites of protease cleavage were identified in the deduced amino acid sequence of mu 1 by determining the amino-terminal sequences of phi proteins: trypsin cleaves between arginine 584 and isoleucine 585, and chymotrypsin cleaves between tyrosine 581 and glycine 582. Findings in this study indicate that sequences in the phi portion of mu 1/mu 1C may participate in the unique functions attributed to ISVPs. Notably, the delta-phi cleavage junction was predicted to be flanked by a pair of long amphipathic alpha-helices. These amphipathic alpha-helices, together with the myristoyl group at the extreme amino terminus of mu 1/mu 1N, are proposed to interact directly with the lipid bilayer of a cell membrane during penetration by mammalian reoviruses. Images PMID:1328674

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions.

PubMed Central

Zillmann, M; Limauro, S E; Goodchild, J

1997-01-01

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core. PMID:9214657

Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.

PubMed

Zhao, F; Stein, D J; Paborji, M; Cash, P W; Root, B J; Wei, Z; Knupp, C J

2001-01-01

BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.

Unusual target site disruption by the rare-cutting HNH restriction endonuclease PacI

PubMed Central

Shen, Betty; Heiter, Daniel F.; Chan, Siu-Hong; Wang, Hua; Xu, Shuang-Yong; Morgan, Richard D.; Wilson, Geoffrey G.; Stoddard, Barry L.

2010-01-01

The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight base pair target recognition sequence 5'-TTAATTAA-3' has been determined to 1.9 Å resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a ββα-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand-cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA basepairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in non-canonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures. PMID:20541511

Proteolytic Processing of Turnip Yellow Mosaic Virus Replication Proteins and Functional Impact on Infectivity▿

PubMed Central

Jakubiec, Anna; Drugeon, Gabrièle; Camborde, Laurent; Jupin, Isabelle

2007-01-01

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA. PMID:17686855

Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

PubMed

Chung, Ivy Yeuk Wah; Paetzel, Mark

2013-05-03

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.

Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris.

PubMed

Ide, Nobuyuki; Masuda, Tetsuya; Kitabatake, Naofumi

2007-11-23

Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.

Detection of site specific glycosylation in proteins using flow cytometry†

PubMed Central

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

Verification of 2A peptide cleavage.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.

Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule.

PubMed

Hackett, C J; Dietzschold, B; Gerhard, W; Ghrist, B; Knorr, R; Gillessen, D; Melchers, F

1983-08-01

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.

qPMS9: An Efficient Algorithm for Quorum Planted Motif Search

NASA Astrophysics Data System (ADS)

Nicolae, Marius; Rajasekaran, Sanguthevar

2015-01-01

Discovering patterns in biological sequences is a crucial problem. For example, the identification of patterns in DNA sequences has resulted in the determination of open reading frames, identification of gene promoter elements, intron/exon splicing sites, and SH RNAs, location of RNA degradation signals, identification of alternative splicing sites, etc. In protein sequences, patterns have led to domain identification, location of protease cleavage sites, identification of signal peptides, protein interactions, determination of protein degradation elements, identification of protein trafficking elements, discovery of short functional motifs, etc. In this paper we focus on the identification of an important class of patterns, namely, motifs. We study the (l, d) motif search problem or Planted Motif Search (PMS). PMS receives as input n strings and two integers l and d. It returns all sequences M of length l that occur in each input string, where each occurrence differs from M in at most d positions. Another formulation is quorum PMS (qPMS), where the motif appears in at least q% of the strings. We introduce qPMS9, a parallel exact qPMS algorithm that offers significant runtime improvements on DNA and protein datasets. qPMS9 solves the challenging DNA (l, d)-instances (28, 12) and (30, 13). The source code is available at https://code.google.com/p/qpms9/.

Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production*

PubMed Central

Zerbe, Philipp; Chiang, Angela; Yuen, Macaire; Hamberger, Björn; Hamberger, Britta; Draper, Jason A.; Britton, Robert; Bohlmann, Jörg

2012-01-01

The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. PMID:22337889

A comparative study of matrix metalloproteinase and aggrecanase mediated release of latent cytokines at arthritic joints.

PubMed

Mullen, Lisa; Adams, Gill; Foster, Julie; Vessillier, Sandrine; Köster, Mario; Hauser, Hansjörg; Layward, Lorna; Gould, David; Chernajovsky, Yuti

2014-09-01

Latent cytokines are engineered by fusing the latency associated peptide (LAP) derived from transforming growth factor-β (TGF-β) with the therapeutic cytokine, in this case interferon-β (IFN-β), via an inflammation-specific matrix metalloproteinase (MMP) cleavage site. To demonstrate latency and specific delivery in vivo and to compare therapeutic efficacy of aggrecanase-mediated release of latent IFN-β in arthritic joints to the original MMP-specific release. Recombinant fusion proteins with MMP, aggrecanase or devoid of cleavage site were expressed in CHO cells, purified and characterised in vitro by Western blotting and anti-viral protection assays. Therapeutic efficacy and half-life were assessed in vivo using the mouse collagen-induced arthritis model (CIA) of rheumatoid arthritis and a model of acute paw inflammation, respectively. Transgenic mice with an IFN-regulated luciferase gene were used to assess latency in vivo and targeted delivery to sites of disease. Efficient localised delivery of IFN-β to inflamed paws, with low levels of systemic delivery, was demonstrated in transgenic mice using latent IFN-β. Engineering of latent IFN-β with an aggrecanase-sensitive cleavage site resulted in efficient cleavage by ADAMTS-4, ADAMTS-5 and synovial fluid from arthritic patients, with an extended half-life similar to the MMP-specific molecule and greater therapeutic efficacy in the CIA model. Latent cytokines require cleavage in vivo for therapeutic efficacy, and they are delivered in a dose dependent fashion only to arthritic joints. The aggrecanase-specific cleavage site is a viable alternative to the MMP cleavage site for the targeting of latent cytokines to arthritic joints. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The Efficiency of Dentin Sialoprotein-Phosphophoryn Processing Is Affected by Mutations Both Flanking and Distant from the Cleavage Site*

PubMed Central

Yang, Robert T.; Lim, Glendale L.; Dong, Zhihong; Lee, Arthur M.; Yee, Colin T.; Fuller, Robert S.; Ritchie, Helena H.

2013-01-01

Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G447↓D448 cleavage site in DSP-PP240 had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P4 to P4′ blocked, impaired, or enhanced DSP-PP240 cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP240 had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP240 significantly modified the amount of PP240 product generated from DSP-PP240 precursor protein cleavage suggests that such mutation may affect the mineralization process. PMID:23297400

The efficiency of dentin sialoprotein-phosphophoryn processing is affected by mutations both flanking and distant from the cleavage site.

PubMed

Yang, Robert T; Lim, Glendale L; Dong, Zhihong; Lee, Arthur M; Yee, Colin T; Fuller, Robert S; Ritchie, Helena H

2013-02-22

Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G(447)↓D(448) cleavage site in DSP-PP(240) had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P(4) to P(4)' blocked, impaired, or enhanced DSP-PP(240) cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP(240) had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP(240) significantly modified the amount of PP(240) product generated from DSP-PP(240) precursor protein cleavage suggests that such mutation may affect the mineralization process.

Antibodies against H3 and H4 histones from the sera of HIV-infected patients catalyze site-specific degradation of these histones.

PubMed

Baranova, Svetlana V; Dmitrenok, Pavel S; Zubkova, Anastasiya D; Ivanisenko, Nikita V; Odintsova, Elena S; Buneva, Valentina N; Nevinsky, Georgy A

2018-02-19

Histones and their posttranslational modified forms play pivotal roles in chromatin functioning and gene transcription. Also, histones are harmful when they enter the intercellular space; their administration to animals results in systemic inflammatory and toxic responses. Autoantibodies having enzymatic activities (abzymes) are the specific feature of several autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical proteases were purified from sera of HIV-infected patients by using several affinity chromatographies. In contrast to known canonical proteases, Abs from HIV-infected patients hydrolyzed exclusively only histones but no other control globular proteins. The H3 and H4 histone cleavage sites by antihistone IgGs were determined by matrix-assisted laser desorption/ionization mass spectrometry for the first time. Two clusters of H3 hydrolysis contain major (↕) and minor (*) cleavage sites: 18-K*Q*LA↕TK*A↕AR*KS↕A*P-30 and 34-G*VK*KPHR*YRPGTVA*L*R-50. H4 histone has only 1 cluster of cleavage sites containing additionally moderate (↓) cleavage sites: 15-A↕KR↕HR↕KVLR↓D*NIQ↓GIT*K-31. Sites of these histones cleavage correspond mainly to their known epitopes. It was surprising that most of the cleavage sites of histones are involved in the interaction with DNA of nucleosome core. Because histones act as damage-associated molecules, abzymes against H3 and H4 can play important role in pathogenesis of AIDs and probably other viral and immune diseases. Copyright © 2018 John Wiley & Sons, Ltd.

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

PubMed

Parua, Pabitra K; Booth, Gregory T; Sansó, Miriam; Benjamin, Bradley; Tanny, Jason C; Lis, John T; Fisher, Robert P

2018-06-13

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity 1 . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination 2 , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat 5 . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing 6 ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression 4 .

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery.

PubMed

Adelman, K; Salmon, B; Baines, J D

2001-03-13

The product of the herpes simplex virus type 1 U(L)28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of U(L)28 in this process, purified U(L)28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for U(L)28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by U(L)28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by U(L)28 protein. To determine the relevance of the observed U(L)28 protein-pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of U(L)28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U(L)28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the U(L)28 protein-pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the U(L)28-encoded component of the herpesvirus cleavage and packaging machinery.

Instability of plasmid DNA sequences: macro and micro evolution of the antibiotic resistance plasmid R6-5.

PubMed

Timmis, K N; Cabello, F; Andrés, I; Nordheim, A; Burkhardt, H J; Cohen, S N

1978-11-16

Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possible other replicons occur more frequently than has hitherto been appreciated. The sequences changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existing in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous, and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site

PubMed Central

Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

2015-01-01

ABSTRACT Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1–PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. PMID:26430213

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site.

PubMed

Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

2015-11-15

Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. © 2015. Published by The Company of Biologists Ltd.

Pathotyping and Phylogenetic Characterization of Newcastle Disease Viruses Isolated in Peru: Defining Two Novel Subgenotypes Within Genotype XII.

PubMed

Chumbe, Ana; Izquierdo-Lara, Ray; Tataje, Luis; Gonzalez, Rosa; Cribillero, Giovana; González, Armando E; Fernández-Díaz, Manolo; Icochea, Eliana

2017-03-01

Infections of poultry with virulent strains of avian paramyxovirus 1 (APMV-1), also known as Newcastle disease viruses (NDVs), cause Newcastle disease (ND). This highly contagious disease affects poultry and many other species of birds worldwide. In countries where the disease is prevalent, constant monitoring and characterization of isolates causing outbreaks are necessary. In this study, we report the results of pathogenicity testing and phylogenetic analyses of seven NDVs isolated from several regions of Peru between 2004 and 2015. Six viruses had intracerebral pathogenicity indices (ICPIs) of between 1.75 and 1.88, corresponding to a velogenic pathotype. The remaining virus had an ICPI of 0.00, corresponding to a lentogenic pathotype. These results were consistent with amino acid sequences at the fusion protein (F) cleavage site. All velogenic isolates had the polybasic amino acid sequence 112 RRQKR↓F 117 at the F cleavage site. Phylogenetic analyses of complete F gene sequences showed that all isolates are classified in class II of APMV-1. The velogenic viruses are classified in genotype XII, while the lentogenic virus is classified in genotype II, closely related to the LaSota vaccine strain. Moreover, tree topology, bootstrap values, and genetic distances observed within genotype XII resulted in the identification of novel subgenotypes XIIa (in South America) and XIIb (in China) and possibly two clades within genotype XIIa. All velogenic Peruvian viruses belonged to subgenotype XIIa. Overall, our results confirm the presence of genotype XII in Peru and suggest that it is the prevalent genotype currently circulating in our country. The phylogenetic characterization of these isolates helps to characterize the evolution of NDV and may help with the development of vaccines specific to our regional necessities.

One precursor, three apolipoproteins: the relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/β-glucan binding protein.

PubMed

Stieb, Stefanie; Roth, Ziv; Dal Magro, Christina; Fischer, Sabine; Butz, Eric; Sagi, Amir; Khalaila, Isam; Lieb, Bernhard; Schenk, Sven; Hoeger, Ulrich

2014-12-01

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the relationship with the large lipid transfer proteins. Two-putative dibasic furin cleavage sites were identified bordering the sequence of the HDL-BGBP. When subjected to mass spectroscopic analyses, tryptic peptides of the large apoprotein of dLp matched the N-terminal part of the precursor, while the peptides obtained for its small apoprotein matched the C-terminal part. Repeating the analysis in the prawn Macrobrachium rosenbergii revealed a similar protein with identical domain architecture suggesting that our findings do not represent an isolated instance. Our results indicate that the above three apolipoproteins (i.e HDL-BGBP and both the large and the small subunit of dLp) are translated as a large precursor. Cleavage at the furin type sites releases two subunits forming a heterodimeric dLP particle, while the remaining part forms an HDL-BGBP whose relationship with other lipoproteins as well as specific functions are yet to be elucidated.

Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro

PubMed Central

Jiang, Shan; Xie, Qing; Zhang, Wei; Zhou, Xia-Qiu; Jin, You-Xin

2005-01-01

AIM: To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis. METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neorpBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with [α-32P]UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37°C in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg2+). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE (containing 8 mol/L urea). RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37°C, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%. CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis. PMID:15996037

Detailed analysis of stem I and its 5' and 3' neighbor regions in the trans-acting HDV ribozyme.

PubMed Central

Nishikawa, F; Roy, M; Fauzi, H; Nishikawa, S

1999-01-01

To determine the stem I structure of the human hepatitis delta virus (HDV) ribozyme, which is related to the substrate sequence in the trans -acting system, we kinetically studied stem I length and sequences. Stem I extension from 7 to 8 or 9 bp caused a loss of activity and a low amount of active complex with 9 bp in the trans -acting system. In a previous report, we presented cleavage in a 6 bp stem I. The observed reaction rates indicate that the original 7 bp stem I is in the most favorable location for catalytic reaction among the possible 6-8 bp stems. To test base specificity, we replaced the original GC-rich sequence in stem I with AU-rich sequences containing six AU or UA base pairs with the natural +1G.U wobble base pair at the cleavage site. The cis -acting AU-rich molecules demonstrated similar catalytic activity to that of the wild-type. In trans -acting molecules, due to stem I instability, reaction efficiency strongly depended on the concentration of the ribozyme-substrate complex and reaction temperature. Multiple turnover was observed at 37 degreesC, strongly suggesting that stem I has no base specificity and more efficient activity can be expected under multiple turnover conditions by substituting several UA or AU base pairs into stem I. We also studied the substrate damaging sequences linked to both ends of stem I for its development in therapeutic applications and confirmed the functions of the unique structure. PMID:9862958

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).

PubMed

Singh, Digvijay; Mallon, John; Poddar, Anustup; Wang, Yanbo; Tippana, Ramreddy; Yang, Olivia; Bailey, Scott; Ha, Taekjip

2018-05-22

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.

PubMed

Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Chen, Ding; Xi, Haitao; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia; Gu, Feng

2017-11-02

Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5'-TTTN-3' protospacer adjacent motif (PAM) at the 5' end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5'-TTN-3' as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells

PubMed Central

Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Chen, Ding; Xi, Haitao; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia

2017-01-01

Abstract Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5′-TTTN-3′ protospacer adjacent motif (PAM) at the 5′ end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5′-TTN-3′ as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications. PMID:28977650

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System

PubMed Central

Meessen-Pinard, Mathieu; Dubé, Mathieu; Day, Robert; Seidah, Nabil G.; Talbot, Pierre J.

2015-01-01

Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G 758 to RRSR↓R 758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies. PMID:26545254

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik

A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appearmore » to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.« less

Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes

PubMed Central

Sheng, Gang; Gogakos, Tasos; Wang, Jiuyu; Zhao, Hongtu; Serganov, Artem; Juranek, Stefan

2017-01-01

Abstract We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6’U7’ and 6’A7’ on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step. We observe a stable alignment for the looped-out 6’N7’ bulge base, which stacks on the unpaired first base of the guide strand, with the looped-out alignment facilitated by weakened Watson–Crick and reversed non-canonical flanking pairs. These structural studies are complemented by cleavage assays that independently monitor the impact of bulges on TtAgo-mediated cleavage reaction. PMID:28911094

Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library

PubMed Central

Marcondes, M.F.M.; Alves, F.M.; Assis, D.M.; Hirata, I.Y.; Juliano, L.; Oliveira, V.; Juliano, M.A.

2015-01-01

The substrate specificity of recombinant human mitochondrial intermediate peptidase (hMIP) using a synthetic support-bound FRET peptide library is presented. The collected fluorescent beads, which contained the hydrolysed peptides generated by hMIP, were sequenced by Edman degradation. The results showed that this peptidase presents a remarkable preference for polar uncharged residues at P1 and P1′ substrate positions: Ser = Gln > Thr at P1 and Ser > Thr at P1′. Non-polar residues were frequent at the substrate P3, P2, P2′ and P3′ positions. Analysis of the predicted MIP processing sites in imported mitochondrial matrix proteins shows these cleavages indeed occur between polar uncharged residues. Previous analysis of these processing sites indicated the importance of positions far from the MIP cleavage site, namely the presence of a hydrophobic residue (Phe or Leu) at P8 and a polar uncharged residue (Ser or Thr) at P5. To evaluate this, additional kinetic analyses were carried out, using fluorogenic substrates synthesized based on the processing sites attributed to MIP. The results described here underscore the importance of the P1 and P1′ substrate positions for the hydrolytic activity of hMIP. The information presented in this work will help in the design of new substrate-based inhibitors for this peptidase. PMID:26082885

A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts.

PubMed

Naveilhan, P; Baudet, C; Jabbour, W; Wion, D

1994-09-01

A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do--namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occurred since the cells involved differentiated during development.

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

PubMed Central

Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

2016-01-01

CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

Structure of the Integral Membrane Protein CAAX Protease Ste24p

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pryor Jr., Edward E.; Horanyi, Peter S.; Clark, Kathleen M.

2012-10-26

Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a -factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavitymore » containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.« less

Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study.

PubMed

Cao, Rui; Wang, T Tiffany; DeMaria, Genevieve; Sheehan, John K; Kesimer, Mehmet

2012-08-03

Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20, which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.

Interaction of Zn(II)bleomycin-A2 and Zn(II)peplomycin with a DNA hairpin containing the 5'-GT-3' binding site in comparison with the 5'-GC-3' binding site studied by NMR spectroscopy.

PubMed

Follett, Shelby E; Ingersoll, Azure D; Murray, Sally A; Reilly, Teresa M; Lehmann, Teresa E

2017-10-01

Bleomycins are a group of glycopeptide antibiotics synthesized by Streptomyces verticillus that are widely used for the treatment of various neoplastic diseases. These antibiotics have the ability to chelate a metal center, mainly Fe(II), and cause site-specific DNA cleavage. Bleomycins are differentiated by their C-terminal regions. Although this antibiotic family is a successful course of treatment for some types of cancers, it is known to cause pulmonary fibrosis. Previous studies have identified that bleomycin-related pulmonary toxicity is linked to the C-terminal region of these drugs. This region has been shown to closely interact with DNA. We examined the binding of Zn(II)peplomycin and Zn(II)bleomycin-A 2 to a DNA hairpin of sequence 5'-CCAGTATTTTTACTGG-3', containing the binding site 5'-GT-3', and compared the results with those obtained from our studies of the same MBLMs bound to a DNA hairpin containing the binding site 5'-GC-3'. We provide evidence that the DNA base sequence has a strong impact in the final structure of the drug-target complex.

Co-circulation of genetically distinct groups of avian paramyxovirus type 1 in pigeon Newcastle disease in Iran.

PubMed

Rezaei Far, A; Peighambari, S M; Pourbakhsh, S A; Ashtari, A; Soltani, M

2017-02-01

Pigeons are considered as one of the major natural reservoirs in the epidemiology of Newcastle disease (ND). In this study, the partial sequence of fusion protein gene of 17 pigeon-origin ND viruses (NDVs) isolated during 2012-2013 in Iran was analysed. Since the studied isolates showed F0 protein cleavage sites compatible with velogenic NDVs, all were considered as virulent NDVs. Two isolates carried 112RRQKRF117 as the cleavage site motif, whereas the rest demonstrated 112KRQKRF117 motif which just recently has been reported among Iranian virulent NDVs. Phylogenetic analysis divided all these diverse isolates in two distinct clusters within class II genotype VI. Based on the partial fusion protein gene sequence, 15 out of 17 isolates showed the highest genetic identity to subgenotype VIb/2 and the other two isolates were placed in a distinct genetic group of genotype VI. Based on recent findings, at least two different sublineages of genotype VI are causing the ND outbreaks in the pigeon population and are circulating simultaneously along with virulent NDVs of genotype VII in various species in Iran. The continuing circulation of a diverse group of virulent NDVs as an enzootic in widespread species such as pigeon can cause outbreaks in commercial poultry flocks and also failure in controlling programmes. Therefore, the constant monitoring and awareness of the virus characteristics should be considered in controlling programmes against ND in Iran.

A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

PubMed Central

Farasat, Iman; Salis, Howard M.

2016-01-01

The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits. PMID:26824432

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

PubMed Central

Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

2017-01-01

Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry.

PubMed

Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R Ogorzalek; Julian, Ryan R; Loo, Joseph A

2015-11-15

The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.

Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

PubMed

Vatter, Heather A; Di, Han; Donaldson, Eric F; Radu, Gertrud U; Maines, Taronna R; Brinton, Margo A

2014-08-01

The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1β, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1β were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites for each PLP1 were confirmed by testing mutant constructs. Several unique features of the SHFV PLP1s were discovered. The SHFV PLP1α catalytic Cys63 is unique among arterivirus PLP1s in being adjacent to an Ala instead of a Trp. Other arterivirus PLP1s cleave only in cis at a single downstream site, but SHFV PLP1γ can cleave at both the downstream nsp1γ-nsp2 and upstream nsp1β-nsp1γ junctions. The three mature nsp1 proteins were produced both in the in vitro reactions and in infected cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase.

PubMed Central

Freemont, P S; Dunbar, B; Fothergill-Gilmore, L A

1988-01-01

The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase, comprising 363 residues, was determined. The sequence was deduced by automated sequencing of CNBr-cleavage, o-iodosobenzoic acid-cleavage, trypsin-digest and staphylococcal-proteinase-digest fragments. Comparison of the sequence with other class I aldolase sequences shows that the mammalian muscle isoenzyme is one of the most highly conserved enzymes known, with only about 2% of the residues changing per 100 million years. Non-mammalian aldolases appear to be evolving at the same rate as other glycolytic enzymes, with about 4% of the residues changing per 100 million years. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Freemont (1988) Biochem. J. 249, 789-793]. PMID:3355497

Analysis of the HLA-DR peptidome from human dendritic cells reveals high affinity repertoires and nonconventional pathways of peptide generation.

PubMed

Ciudad, M Teresa; Sorvillo, Nicoletta; van Alphen, Floris P; Catalán, Diego; Meijer, Alexander B; Voorberg, Jan; Jaraquemada, Dolores

2017-01-01

Dendritic cells (DCs) are the major professional APCs of the immune system; however, their MHC-II-associated peptide repertoires have been hard to analyze, mostly because of their scarce presence in blood and tissues. In vitro matured human monocyte-derived DCs (MoDCs) are widely used as professional APCs in experimental systems. In this work, we have applied mass spectrometry to identify the HLA-DR-associated self-peptide repertoires from small numbers of mature MoDCs (∼5 × 10 6 cells), derived from 7 different donors. Repertoires of 9 different HLA-DR alleles were defined from analysis of 1319 peptides, showing the expected characteristics of MHC-II-associated peptides. Most peptides identified were predicted high binders for their respective allele, formed nested sets, and belonged to endo-lysosomal pathway-degraded proteins. Approximately 20% of the peptides were derived from cytosolic and nuclear proteins, a recurrent finding in HLA-DR peptide repertoires. Of interest, most of these peptides corresponded to single sequences, did not form nested sets, and were located at the C terminus of the parental protein, which suggested alternative processing. Analysis of cleavage patterns for terminal peptides predominantly showed aspartic acid before the cleavage site of both C- and N-terminal peptides and proline immediately after the cleavage site in C-terminal peptides. Proline was also frequent next to the cut sites of internal peptides. These data provide new insights into the Ag processing capabilities of DCs. The relevance of these processing pathways and their contribution to response to infection, tolerance induction, or autoimmunity deserve further analysis. © Society for Leukocyte Biology.

Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes.

PubMed

Kawaguchi, Mari; Yasumasu, Shigeki; Shimizu, Akio; Sano, Kaori; Iuchi, Ichiro; Nishida, Mutsumi

2010-12-01

We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved. © 2010 The Authors Journal compilation © 2010 FEBS.

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12.

PubMed

Claverie-Martin, F; Diaz-Torres, M R; Kushner, S R

1987-01-01

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.

Footprinting reveals that nogalamycin and actinomycin shuffle between DNA binding sites.

PubMed Central

Fox, K R; Waring, M J

1986-01-01

The hypothesis that sequence-selective DNA-binding antibiotics locate their preferred binding sites by a process involving migration from nonspecific sites has been tested by footprinting with DNAase I. Footprinting patterns on the tyrT DNA fragment produced by nogalamycin and actinomycin change with time after mixing the antibiotic with the DNA. Sites of protection as well as enhanced cleavage are seen to develop in a fashion which is both temperature and concentration-dependent. At certain sites cutting is transiently enhanced, then blocked. Limited evidence for slow reaction with echinomycin and mithramycin is presented, but the kinetics of footprinting with daunomycin and distamycin appear instantaneous. The feasibility of adducing direct evidence for shuffling by footprinting seems to be governed by slow dissociation of the antibiotic-DNA complex. It may also be dependent upon the mode of binding, be it intercalative or non-intercalative in character. Images PMID:2421246

The maturation inhibitor bevirimat (PA-457) can be active in patients carrying HIV type-1 non-B subtypes and recombinants.

PubMed

Yebra, Gonzalo; Holguín, Africa

2008-01-01

Bevirimat (PA-457) is the first candidate of a new family of antiretroviral drugs, the maturation inhibitors. Its action is based on disruption of the protease cleavage of the Gag precursor region. Six resistance mutations have been described and analysed in virus from both treatment-naive and protease inhibitor (PI)-experienced patients, but only in the subtype B of HIV type-1 (HIV-1) virus. Thus, genotypic resistance in non-B subtypes still requires analysis. HIV-1 sequences of different subtypes (54 B, 81 non-B and recombinants) were analysed for the presence of resistance mutations to bevirimat, located within the capsid (CA) protein and spacer peptide 1 (SP1) cleavage site. No resistance mutations were found, although polymorphisms appeared in some CA-SP1 residues. The C-terminal CA protein and the N-terminal SP1 presented high conservation, whereas C-terminal SP1 was highly variable in sequence and length, with unknown influence in resistance acquisition. The results of the present study confirm an absolute conservation of the residues involved in bevirimat in vitro resistance in a large panel of HIV-1 subtypes and recombinants from both treatment-naive and PI-experienced patients. Treatment alone seemed to increase the polymorphisms account in CRF02_AG recombinant sequences; however, the influence of natural polymorphisms needs to be explored.

An Internal Signal Sequence Directs Intramembrane Proteolysis of a Cellular Immunoglobulin Domain Protein*S⃞

PubMed Central

Robakis, Thalia; Bak, Beata; Lin, Shu-huei; Bernard, Daniel J.; Scheiffele, Peter

2008-01-01

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins. PMID:18981173

Kinetics of hairpin ribozyme cleavage in yeast.

PubMed Central

Donahue, C P; Fedor, M J

1997-01-01

Hairpin ribozymes catalyze a self-cleavage reaction that provides a simple model for quantitative analyses of intracellular mechanisms of RNA catalysis. Decay rates of chimeric mRNAs containing self-cleaving ribozymes give a direct measure of intracellular cleavage kinetics in yeast. Intracellular ribozyme-mediated cleavage occurs at similar rates and shows similar inhibition by ribozyme mutations as ribozyme-mediated reactions in vitro, but only when ribozymes are located in a favorable mRNA sequence context. The impact of cleavage on mRNA abundance is shown to depend directly on intrinsic mRNA stability. Surprisingly, cleavage products are no more labile than uncleaved mRNAs despite the loss of terminal cap structures or poly (A). PMID:9292496

Molecular characteristics of insect vitellogenins and vitellogenin receptors.

PubMed

Sappington, T W; Raikhel, A S

1998-01-01

The recent cloning and sequencing of several insect vitellogenins (Vg), the major yolk protein precursor of most oviparous animals, and the mosquito Vg receptor (VgR) has brought the study of insect vitellogenesis to a new plane. Insect Vgs are homologous to nematode and vertebrate Vgs. All but one of the insect Vgs for which we know the primary structure are cleaved into two subunits at a site [(R/K)X(R/K)R or RXXR with an adjacent beta-turn] recognized by subtilisin-like proprotein convertases. In four of the Vgs, the cleavage site is near the N-terminus, but in one insect species, it is near the C-terminus of the Vg precursor. Multiple alignments of these Vg sequences indicate that the variation in cleavage location has not arisen through exon shuffling, but through local modifications of the amino acid sequences. A wasp Vg precursor is not cleaved, apparently because the sequence at the presumed ancestral cleavage site has been mutated from RXRR to LYRR and is no longer recognized by convertases. Some insect Vgs contain polyserine domains which are reminiscent of, but not homologous to, the phosvitin domain in vertebrate Vgs. The sequence of the mosquito VgR revealed that it is a member of the low-density lipoprotein receptor (LDLR) family. Though resembling chicken and frog VgRs, which are also members of the LDLR family, it is twice as big, carrying two clusters of cysteine-rich complement-type (Class A) repeats (implicated in ligand-binding) instead of one like vertebrate VgRs and LDLRs. It is very similar in sequence and domain arrangement to the Drosophila yolk protein receptor (YPR), despite a non-vitellogenin ligand for the latter. Though vertebrate VgRs, insect VgR/YPRs, and LDLR-related proteins/megalins all accommodate one cluster of eight Class A repeats, fingerprint analysis of the repeats in these clusters indicate they are not directly homologous with one another, but have undergone differing histories of duplications, deletions, and exon shuffling so that their apparent similarity is superficial. The so-called epidermal growth factor precursor region contains two types of motifs (cysteine-rich Class B repeats and YWXD repeats) which occur independently of one another in diverse proteins, and are often involved in protein-protein interactions, suggesting that they potentially are involved in dimerization of VgRs and other LDLR-family proteins. Like the LDLR, but unlike vertebrate VgRs and the Drosophila YPR, the mosquito VgR contains a putative O-linked sugar region on the extra-cellular side of the transmembrane domain. Its function is unclear, but may protect the receptor from membrane-bound proteases. The cytoplasmic tail of insect VgR/YPRs contains a di-leucine (or leucine-isoleucine) internalization signal, unlike the tight-turn tyrosine motif of other LDLR-family proteins. The importance of understanding the details of yolk protein uptake by oocytes lies in its potential for exploitation in novel insect control strategies, and the molecular characterization of the proteins involved has made the development of such strategies a realistic possibility.

A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

NASA Astrophysics Data System (ADS)

Poston, Chloe N.; Higgs, Richard E.; You, Jinsam; Gelfanova, Valentina; Hale, John E.; Knierman, Michael D.; Siegel, Robert; Gutierrez, Jesus A.

2014-07-01

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

A quantitative tool to distinguish isobaric leucine and isoleucine residues for mass spectrometry-based de novo monoclonal antibody sequencing.

PubMed

Poston, Chloe N; Higgs, Richard E; You, Jinsam; Gelfanova, Valentina; Hale, John E; Knierman, Michael D; Siegel, Robert; Gutierrez, Jesus A

2014-07-01

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

The roles of SSU processome components and surveillance factors in the initial processing of human ribosomal RNA

PubMed Central

Sloan, Katherine E.; Bohnsack, Markus T.; Schneider, Claudia; Watkins, Nicholas J.

2014-01-01

During eukaryotic ribosome biogenesis, three of the mature ribosomal (r)RNAs are released from a single precursor transcript (pre-rRNA) by an ordered series of endonucleolytic cleavages and exonucleolytic processing steps. Production of the 18S rRNA requires the removal of the 5′ external transcribed spacer (5′ETS) by endonucleolytic cleavages at sites A0 and A1/site 1. In metazoans, an additional cleavage in the 5′ETS, at site A′, upstream of A0, has also been reported. Here, we have investigated how A′ processing is coordinated with assembly of the early preribosomal complex. We find that only the tUTP (UTP-A) complex is critical for A′ cleavage, while components of the bUTP (UTP-B) and U3 snoRNP are important, but not essential, for efficient processing at this site. All other factors involved in the early stages of 18S rRNA processing that were tested here function downstream from this processing step. Interestingly, we show that the RNA surveillance factors XRN2 and MTR4 are also involved in A′ cleavage in humans. A′ cleavage is largely bypassed when XRN2 is depleted, and we also discover that A′ cleavage is not always the initial processing event in all cell types. Together, our data suggest that A′ cleavage is not a prerequisite for downstream pre-rRNA processing steps and may, in fact, represent a quality control step for initial pre-rRNA transcripts. Furthermore, we show that components of the RNA surveillance machinery, including the exosome and TRAMP complexes, also play key roles in the recycling of excised spacer fragments and degradation of aberrant pre-rRNAs in human cells. PMID:24550520

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

PubMed Central

Lott, William B.; Pontius, Brian W.; von Hippel, Peter H.

1998-01-01

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 μM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and >50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and >37.5 μM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2′-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5′-oxygen in the transition state. We suggest structural reasons why the Mg2+–La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction. PMID:9435228

Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

PubMed

Chaumont, F; Silva Filho, M de C; Thomas, D; Leterme, S; Boutry, M

1994-02-01

The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

Expanding proteome coverage with orthogonal-specificity α-Lytic proteases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.

2014-03-01

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage by cleavage at sequences complimentary to trypsin may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type alpha-lytic protease (WaLP), and an active site mutant of WaLP, M190A alpha-lytic protease (MaLP). We assess several relevant factors including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. Bymore » combining data from separate digestions with trypsin, LysC, WaLP, and MaLP, proteome coverage was increased 101% compared to trypsin digestion alone. To demonstrate how the gained sequence coverage can access additional PTM information, we show identification of a number of novel phosphorylation sites in the S. pombe proteome and include an illustrative example from the protein MPD2, wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.« less

Cleavage of the Drosophila seminal protein Acp36DE in mated females enhances its sperm storage activity.

PubMed

Avila, Frank W; Wolfner, Mariana F

2017-08-01

Sperm storage in the mated female reproductive tract (RT) is required for optimal fertility in numerous species with internal fertilization. In Drosophila melanogaster, sperm storage is dependent on female receipt of seminal fluid proteins (SFPs) during mating. The seminal fluid protein Acp36DE is necessary for the accumulation of sperm into storage. In the female RT, Acp36DE localizes to the anterior mating plug and also to a site in the common oviduct, potentially "corralling" sperm near the entry sites into the storage organs. Genetic studies showed that Acp36DE is also required for a series of conformational changes of the uterus that begin at the onset of mating and are hypothesized to move sperm towards the entry sites of the sperm storage organs. After Acp36DE is transferred to the female RT, the protein is cleaved by the astacin-metalloprotease Semp1. However, the effect of this cleavage on Acp36DE's function in sperm accumulation into storage is unknown. We used mass spectrometry to identify the single cleavage site in Acp36DE. We then mutated this site and tested the effects on sperm storage. Mutations of Acp36DE's cleavage site that slowed or prevented cleavage of the protein slowed the accumulation of sperm into storage, although they did not affect uterine conformational changes in mated females. Moreover, the N-terminal cleavage product of Acp36DE was sufficient to mediate sperm accumulation in storage, and it did so faster than versions of Acp36DE that could not be cleaved or were only cleaved slowly. These results suggest that cleavage of Acp36E may increase the number of bioactive molecules within the female RT, a mechanism similar to that hypothesized for Semp1's other substrate, the seminal fluid protein ovulin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural basis of UGUA recognition by the Nudix protein CFIm25 and implications for a regulatory role in mRNA 3′ processing

PubMed Central

Yang, Qin; Gilmartin, Gregory M.; Doublié, Sylvie

2010-01-01

Human Cleavage Factor Im (CFIm) is an essential component of the pre-mRNA 3′ processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFIm25) of the CFIm complex possesses a characteristic α/β/α Nudix fold, CFIm25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFIm25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFIm25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson–Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap4A (diadenosine tetraphosphate) by CFIm25 suggests a potential role for small molecules in the regulation of mRNA 3′ processing. PMID:20479262

Structural basis of UGUA recognition by the Nudix protein CFI(m)25 and implications for a regulatory role in mRNA 3' processing.

PubMed

Yang, Qin; Gilmartin, Gregory M; Doublié, Sylvie

2010-06-01

Human Cleavage Factor Im (CFI(m)) is an essential component of the pre-mRNA 3' processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI(m)25) of the CFI(m) complex possesses a characteristic alpha/beta/alpha Nudix fold, CFI(m)25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI(m)25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI(m)25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap(4)A (diadenosine tetraphosphate) by CFI(m)25 suggests a potential role for small molecules in the regulation of mRNA 3' processing.

Modeling the full length HIV-1 Gag polyprotein reveals the role of its p6 subunit in viral maturation and the effect of non-cleavage site mutations in protease drug resistance.

PubMed

Su, Chinh Tran-To; Kwoh, Chee-Keong; Verma, Chandra Shekhar; Gan, Samuel Ken-En

2017-12-27

HIV polyprotein Gag is increasingly found to contribute to protease inhibitor resistance. Despite its role in viral maturation and in developing drug resistance, there remain gaps in the knowledge of the role of certain Gag subunits (e.g. p6), and that of non-cleavage mutations in drug resistance. As p6 is flexible, it poses a problem for structural experiments, and is hence often omitted in experimental Gag structural studies. Nonetheless, as p6 is an indispensable component for viral assembly and maturation, we have modeled the full length Gag structure based on several experimentally determined constraints and studied its structural dynamics. Our findings suggest that p6 can mechanistically modulate Gag conformations. In addition, the full length Gag model reveals that allosteric communication between the non-cleavage site mutations and the first Gag cleavage site could possibly result in protease drug resistance, particularly in the absence of mutations in Gag cleavage sites. Our study provides a mechanistic understanding to the structural dynamics of HIV-1 Gag, and also proposes p6 as a possible drug target in anti-HIV therapy.

Synthesis and DNA interaction of a mixed proflavine-phenanthroline Tröger base.

PubMed

Baldeyrou, Brigitte; Tardy, Christelle; Bailly, Christian; Colson, Pierre; Houssier, Claude; Charmantray, Franck; Demeunynck, Martine

2002-04-01

We report the synthesis of an asymmetric Tröger base containing the two well characterised DNA binding chromophores, proflavine and phenanthroline. The mode of interaction of the hybrid molecule was investigated by circular and linear dichroism experiments and a biochemical assay using DNA topoisomerase I. The data are compatible with a model in which the proflavine moiety intercalates between DNA base pairs and the phenanthroline ring occupies the DNA groove. DNase I cleavage experiments were carried out to investigate the sequence preference of the hybrid ligand and a well resolved footprint was detected at a site encompassing two adjacent 5'-GTC.5-GAC triplets. The sequence preference of the asymmetric molecule is compared to that of the symmetric analogues.

The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

PubMed Central

Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

PubMed

Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties

PubMed Central

Somyoonsap, Peechapack; Kitpreechavanich, Vichein

2013-01-01

A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71 kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg2+ as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease. PMID:25937959

Pre-lithification tectonic foliation development in a clastic sedimentary rock sequence from SW Ireland

NASA Astrophysics Data System (ADS)

Meere, Patrick; Mulchrone, Kieran; McCarthy, David

2017-04-01

The current orthodoxy regarding the development of regionally developed penetrative tectonic cleavage fabrics in sedimentary rocks is that it postdates lithification of those rocks. It is well established that fabric development under these circumstances is achieved by a combination of grain rigid body rotation, crystal-plastic deformation and pressure solution. The latter is believed to be the primary mechanism responsible for the domainal nature of cleavage development commonly observed in low grade metamorphic rocks. While there have been advocates for the development of tectonic cleavages before host rock lithification these are currently viewed as essentially local aberrations without regional significance. In this study we combine new field observations with strain analysis, element mapping and modelling to characterise Acadian (>50%) crustal shortening in a Devonian clastic sedimentary sequence from the Dingle Peninsula of south west Ireland. Fabrics in these rocks reflect significant levels of tectonic shortening are a product of grain translation, rigid body rotation and repacking of intra- and extra-formational clasts during deformation of an unconsolidated clastic sedimentary sequence. There is an absence of the expected domainal cleavage structure and intra-clast deformation expected with conventional cleavage formation. This study requires geologists to consider the possibility such a mechanism contributing to tectonic strain in a wide range of geological settings and to look again at field evidence that indicates early sediment mobility during deformation.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

PubMed Central

Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

2015-01-01

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

Processing of mammalian preprogastrin-releasing peptide.

PubMed

Reeve, J R; Cuttitta, F; Vigna, S R; Shively, J E; Walsh, J H

1988-01-01

The processing of preprogastrin-releasing peptide in mammalian tissues and in cultured cells takes place at discrete sites (Figure 6). Signal peptidase cleaves away the signal peptide from the amino terminus of gastrin-releasing peptide. An exopeptidase activity may remove dipeptides from the amino terminus. The amidation site (not shown in Fig. 6; see Fig. 2) has the same general sequence (Gly-Lys-Lys) seen for other amidated peptides. Cleavage after single basic residues yields gene-related products from Form I or II preproGRP. A unique non-basic cleavage yields a gene-related product from Form III preproGRP. The processing that occurs to form GRP, GRP, and GRP gene-related peptides is shown in Figure 7. ProGRP is cleaved by a series of enzymes to form GRP with an amidated carboxyl-terminal methionine (indicated by an asterisk in Fig. 7). GRP is cleaved to form the decapeptide GRP. The carboxyl-terminal flanking peptides of all three mRNA translation products are cleaved to form several gastrin-releasing peptide gene-related products. Knowledge of the processing of gastrin-releasing peptide and its gene-related products will allow synthesis of duplicates of the stored forms of these peptides, which can then be used for biological testing.

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

PubMed Central

Grawunder, U; Lieber, M R

1997-01-01

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining phase of the V(D)J recombination reaction. PMID:9060432

Distinct oxidative cleavage and modification of bovine [Cu-Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule

PubMed Central

Uehara, Hiroshi; Luo, Shen; Aryal, Baikuntha; Levine, Rodney L.; Rao, V. Ashutosh

2016-01-01

We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu-Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2 kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5 Angstrom from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction. PMID:26872685

Distinct oxidative cleavage and modification of bovine [Cu- Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule.

PubMed

Uehara, Hiroshi; Luo, Shen; Aryal, Baikuntha; Levine, Rodney L; Rao, V Ashutosh

2016-05-01

We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu-Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5Å from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction. Published by Elsevier Inc.

Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

PubMed

Bozeman, Trevor C; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J; Zaleski, Paul A; Wilson, W David; Hecht, Sidney M

2012-10-31

Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.

PubMed

Li, Lixin; Piatek, Marek J; Atef, Ahmed; Piatek, Agnieszka; Wibowo, Anjar; Fang, Xiaoyun; Sabir, J S M; Zhu, Jian-Kang; Mahfouz, Magdy M

2012-03-01

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

Surface Diversity in Mycoplasma agalactiae Is Driven by Site-Specific DNA Inversions within the vpma Multigene Locus

PubMed Central

Glew, Michelle D.; Marenda, Marc; Rosengarten, Renate; Citti, Christine

2002-01-01

The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5′ untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5′ untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the λ integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae. PMID:12374833

BplI, a new BcgI-like restriction endonuclease, which recognizes a symmetric sequence.

PubMed Central

Vitkute, J; Maneliene, Z; Petrusyte, M; Janulaitis, A

1997-01-01

Bcg I and Bcg I-like restriction endonucleases cleave double stranded DNA specifically on both sides of their asymmetric recognition sequences which are interrupted by several ambiguous base pairs. Their heterosubunit structure, bifunctionality and stimulation by AdoMet make them different from other classified restriction enzymes. Here we report on a new Bcg I-like restriction endonuclease, Bpl I from Bacillus pumilus , which in contrast to all other Bcg I-like enzymes, recognizes a symmetric interrupted sequence, and which, like Bcg I, cleaves double stranded DNA upstream and downstream of its recognition sequence (8/13)GAGN5CTC(13/8). Like Bcg I, Bpl I is a bifunctional enzyme revealing both DNA cleavage and methyltransferase activities. There are two polypeptides in the homogeneous preparation of Bpl I with molecular masses of approximately 74 and 37 kDa. The sizes of the Bpl I subunits are close to those of Bcg I, but the proportion 1:1 in the final preparation is different from that of 2:1 in Bcg I. Low activity observed with Mg2+increases >100-fold in the presence of AdoMet. Even with AdoMet though, specific cleavage is incomplete. S -adenosylhomocysteine (AdoHcy) or sinefungin can replace AdoMet in the cleavage reaction. AdoHcy activated Bpl I yields complete cleavage of DNA. PMID:9358150

A peptide-based approach to evaluate the adaptability of influenza A virus to humans based on its hemagglutinin proteolytic cleavage site

PubMed Central

Straus, Marco R.; Whittaker, Gary R.

2017-01-01

Cleavage activation of the hemagglutinin (HA) protein by host proteases is a crucial step in the infection process of influenza A viruses (IAV). However, IAV exists in eighteen different HA subtypes in nature and their cleavage sites vary considerably. There is uncertainty regarding which specific proteases activate a given HA in the human respiratory tract. Understanding the relationship between different HA subtypes and human-specific proteases will be valuable in assessing the pandemic potential of circulating viruses. Here we utilized fluorogenic peptides mimicking the HA cleavage motif of representative IAV strains causing disease in humans or of zoonotic/pandemic potential and tested them with a range of proteases known to be present in the human respiratory tract. Our results show that peptides from the H1, H2 and H3 subtypes are cleaved efficiently by a wide range of proteases including trypsin, matriptase, human airway tryptase (HAT), kallikrein-related peptidases 5 (KLK5) and 12 (KLK12) and plasmin. Regarding IAVs currently of concern for human adaptation, cleavage site peptides from H10 viruses showed very limited cleavage by respiratory tract proteases. Peptide mimics from H6 viruses showed broader cleavage by respiratory tract proteases, while H5, H7 and H9 subtypes showed variable cleavage; particularly matriptase appeared to be a key protease capable of activating IAVs. We also tested HA substrate specificity of Factor Xa, a protease required for HA cleavage in chicken embryos and relevant for influenza virus production in eggs. Overall our data provide novel tool allowing the assessment of human adaptation of IAV HA subtypes. PMID:28358853

Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop.

PubMed

Kettunen, R; Tyystjärvi, E; Aro, E M

1996-08-01

Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. this cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves.

BIOPEP database and other programs for processing bioactive peptide sequences.

PubMed

Minkiewicz, Piotr; Dziuba, Jerzy; Iwaniak, Anna; Dziuba, Marta; Darewicz, Małgorzata

2008-01-01

This review presents the potential for application of computational tools in peptide science based on a sample BIOPEP database and program as well as other programs and databases available via the World Wide Web. The BIOPEP application contains a database of biologically active peptide sequences and a program enabling construction of profiles of the potential biological activity of protein fragments, calculation of quantitative descriptors as measures of the value of proteins as potential precursors of bioactive peptides, and prediction of bonds susceptible to hydrolysis by endopeptidases in a protein chain. Other bioactive and allergenic peptide sequence databases are also presented. Programs enabling the construction of binary and multiple alignments between peptide sequences, the construction of sequence motifs attributed to a given type of bioactivity, searching for potential precursors of bioactive peptides, and the prediction of sites susceptible to proteolytic cleavage in protein chains are available via the Internet as are other approaches concerning secondary structure prediction and calculation of physicochemical features based on amino acid sequence. Programs for prediction of allergenic and toxic properties have also been developed. This review explores the possibilities of cooperation between various programs.

Evolutionary dynamics of Newcastle disease virus

USGS Publications Warehouse

Miller, P.J.; Kim, L.M.; Ip, Hon S.; Afonso, C.L.

2009-01-01

A comprehensive dataset of NDV genome sequences was evaluated using bioinformatics to characterize the evolutionary forces affecting NDV genomes. Despite evidence of recombination in most genes, only one event in the fusion gene of genotype V viruses produced evolutionarily viable progenies. The codon-associated rate of change for the six NDV proteins revealed that the highest rate of change occurred at the fusion protein. All proteins were under strong purifying (negative) selection; the fusion protein displayed the highest number of amino acids under positive selection. Regardless of the phylogenetic grouping or the level of virulence, the cleavage site motif was highly conserved implying that mutations at this site that result in changes of virulence may not be favored. The coding sequence of the fusion gene and the genomes of viruses from wild birds displayed higher yearly rates of change in virulent viruses than in viruses of low virulence, suggesting that an increase in virulence may accelerate the rate of NDV evolution. ?? 2009 Elsevier Inc.

CC-1065 functional analogues possessing different electron-withdrawing substituents and leaving groups: synthesis, kinetics, and sequence specificity of reaction with DNA and biological evaluation.

PubMed

Wang, Y; Gupta, R; Huang, L; Lown, J W

1993-12-24

Antitumor agent CC-1065 functional analogues possessing different electron-withdrawing substituents and leaving groups have been synthesized. The extent and the relative rates of DNA cleavage following alkylation by these CPI structures and thermal treatment were determined independently by an ethidium binding assay and by agarose gel electrophoresis experiments. The anticipated preferential covalent binding to adenine sites within the minor groove was confirmed by sequencing determination of selected agents on high-resolution gels. Certain of the synthetic agents, unlike CC-1065, also bind covalently to G sites with weaker intensity. The cytotoxicities of these compounds were also determined against KB cells in vitro. Compounds bearing a bromo or nitro group in the benzene ring and a methylsulfonyl as a leaving group are 10 and 5 times more potent than their unsubstituted counterparts, respectively. Compounds bearing a methylsulfonyl as a leaving group are more potent than those bearing a chlorine.

Remarkable alkaline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution

PubMed Central

Minakuchi, Kazunobu; Murata, Dai; Okubo, Yuji; Nakano, Yoshiyuki; Yoshida, Shinichi

2013-01-01

Protein A affinity chromatography is the standard purification process for the capture of therapeutic antibodies. The individual IgG-binding domains of protein A (E, D, A, B, C) have highly homologous amino acid sequences. From a previous report, it has been assumed that the C domain has superior resistance to alkaline conditions compared to the other domains. We investigated several properties of the C domain as an IgG-Fc capture ligand. Based on cleavage site analysis of a recombinant protein A using a protein sequencer, the C domain was found to be the only domain to have neither of the potential alkaline cleavage sites. Circular dichroism (CD) analysis also indicated that the C domain has good physicochemical stability. Additionally, we evaluated the amino acid substitutions at the Gly-29 position of the C domain, as the Z domain (an artificial B domain) acquired alkaline resistance through a G29A mutation. The G29A mutation proved to increase the alkaline resistance of the C domain, based on BIACORE analysis, although the improvement was significantly smaller than that observed for the B domain. Interestingly, a number of other amino acid mutations at the same position increased alkaline resistance more than did the G29A mutation. This result supports the notion that even a single mutation on the originally alkali-stable C domain would improve its alkaline stability. An engineered protein A based on this C domain is expected to show remarkable performance as an affinity ligand for immunoglobulin. PMID:23868198

Transformable Rhodobacter strains, method for producing transformable Rhodobacter strains

DOEpatents

Laible, Philip D.; Hanson, Deborah K.

2018-05-08

The invention provides an organism for expressing foreign DNA, the organism engineered to accept standard DNA carriers. The genome of the organism codes for intracytoplasmic membranes and features an interruption in at least one of the genes coding for restriction enzymes. Further provided is a system for producing biological materials comprising: selecting a vehicle to carry DNA which codes for the biological materials; determining sites on the vehicle's DNA sequence susceptible to restriction enzyme cleavage; choosing an organism to accept the vehicle based on that organism not acting upon at least one of said vehicle's sites; engineering said vehicle to contain said DNA; thereby creating a synthetic vector; and causing the synthetic vector to enter the organism so as cause expression of said DNA.

Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage.

PubMed

Guan, Su Hua; Belsham, Graham J

2017-04-01

Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

PubMed Central

Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

2017-01-01

The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A. PMID:28052131

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deutscher, J.; Pevec, B.; Beyreuther, K.

1986-10-21

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system,more » HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.« less

A novel eight amino acid insertion contributes to the hemagglutinin cleavability and the virulence of a highly pathogenic avian influenza A (H7N3) virus in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Xiangjie; Belser, Jessica A.; Tumpey, Terrence M., E-mail: tft9@cdc.gov

In 2012, an avian influenza A H7N3 (A/Mexico/InDRE7218/2012; Mx/7218) virus was responsible for two confirmed cases of human infection and led to the death or culling of more than 22 million chickens in Jalisco, Mexico. Interestingly, this virus acquired an 8-amino acid (aa)-insertion (..PENPK-DRKSRHRR-TR/GLF) near the hemagglutinin (HA) cleavage site by nonhomologous recombination with host rRNA. It remains unclear which specific residues at the cleavage site contribute to the virulence of H7N3 viruses in mammals. Using loss-of-function approaches, we generated a series of cleavage site mutant viruses by reverse genetics and characterized the viruses in vitro and in vivo. Wemore » found that the 8-aa insertion and the arginine at position P4 of the Mx/7218 HA cleavage site are essential for intracellular HA cleavage in 293T cells, but have no effect on the pH of membrane fusion. However, we identified a role for the histidine residue at P5 position in viral fusion pH. In mice, the 8-aa insertion is required for Mx/7218 virus virulence; however, the basic residues upstream of the P4 position are dispensable for virulence. Overall, our study provides the first line of evidence that the insertion in the Mx/7218 virus HA cleavage site confers its intracellular cleavability, and consequently contributes to enhanced virulence in mice. - Highlights: • An avian influenza H7N3 virus acquired a unique 8-amino acid (aa) insertion. • The role of specific basic residues in the HA insertion in viral pathogenesis was determined. • In mice, the 8-aa insertion is required for H7N3 virus virulence. • The R residue at position P4 is essential for HA intracellular cleavage and virus virulence.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kem, W.; Dunn, B.; Parten, B.

A 5000 dalton polypeptide neurotoxin (Sh-NI) purified by G50 Sephadex, P-cellulose, and SP-Sephadex chromatography was homogeneous by isoelectric focusing. Sh-NI was highly toxic to crayfish (LD/sub 50/ 0.6 ..mu..g/kg) but without effect upon mice at 15,000 ..mu..g/kg (i.p. injection). The reduced, /sup 3/H-carboxymethylated toxin and its fragments were subjected to automatic Edman degradation and the resulting PTH-amino acids were identified by HPLC, back hydrolysis, and scintillation counting. Peptides resulting from proteolytic (clostripain, staphylococcal protease) and chemical (tryptophan) cleavage were sequenced. The sequence is: AACKCDDEGPDIRTAPLTGTVDLGSCNAGWEKCASYYTIIADCCRKKK. This sequence differs considerably from the homologous Anemonia and Anthopleura toxins; many of the identical residuesmore » (6 half-cystines, G9, P10, R13, G19, G29, W30) are probably critical for folding rather than receptor recognition. However, the Sh-NI sequence closely resembles Radioanthus macrodactylus neurotoxin III and r. paumotensis II. The authors propose that Sh-NI and related Radioanthus toxins act upon a different site on the sodium channel.« less

No evidence for a role of modified live virus vaccines in the emergence of canine parvovirus.

PubMed

Truyen, U; Geissler, K; Parrish, C R; Hermanns, W; Siegl, G

1998-05-01

In this study the early evolution and potential origins of canine parvovirus (CPV) were examined. We cloned and sequenced the VP2 capsid protein genes of three German CPV strains isolated in 1979-1980, as well as two feline panleukopenia virus (FPV) vaccine viruses that were previously shown to have some restriction enzyme cleavage sites in common with CPV. Other partial VP2 gene sequences were obtained by amplifying CPV DNA from paraffin-embedded tissues of dogs which were early parvovirus disease cases in Germany in 1978-1979. Sequences were analysed with respect to their evolutionary relationships to other CPV and FPV isolates. Those analyses did not support the hypothesis that CPV emerged as a variant of an FPV vaccine virus. Neither did they reveal ancestral sequences among the very early CPV isolates examined. Other possible sources for the origin of CPV are examined, including the involvement of viruses from wild carnivores.

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

PubMed

De Marco, L; Mazzucato, M; Masotti, A; Ruggeri, Z M

1994-03-04

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

A transcriptional blueprint for a spiral-cleaving embryo.

PubMed

Chou, Hsien-Chao; Pruitt, Margaret M; Bastin, Benjamin R; Schneider, Stephan Q

2016-08-05

The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.

Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome

PubMed Central

Fowler, Kyle R.; Sasaki, Mariko; Milman, Neta

2014-01-01

Fission yeast Rec12 (Spo11 homolog) initiates meiotic recombination by forming developmentally programmed DNA double-strand breaks (DSBs). DSB distributions influence patterns of heredity and genome evolution, but the basis of the highly nonrandom choice of Rec12 cleavage sites is poorly understood, largely because available maps are of relatively low resolution and sensitivity. Here, we determined DSBs genome-wide at near-nucleotide resolution by sequencing the oligonucleotides attached to Rec12 following DNA cleavage. The single oligonucleotide size class allowed us to deeply sample all break events. We find strong evidence across the genome for differential DSB repair accounting for crossover invariance (constant cM/kb in spite of DSB hotspots). Surprisingly, about half of all crossovers occur in regions where DSBs occur at low frequency and are widely dispersed in location from cell to cell. These previously undetected, low-level DSBs thus play an outsized and crucial role in meiosis. We further find that the influence of underlying nucleotide sequence and chromosomal architecture differs in multiple ways from that in budding yeast. DSBs are not strongly restricted to nucleosome-depleted regions, as they are in budding yeast, but are nevertheless spatially influenced by chromatin structure. Our analyses demonstrate that evolutionarily fluid factors contribute to crossover initiation and regulation. PMID:25024163

Riboswitch-based sensor in low optical background

NASA Astrophysics Data System (ADS)

Harbaugh, Svetlana V.; Davidson, Molly E.; Chushak, Yaroslav G.; Kelley-Loughnane, Nancy; Stone, Morley O.

2008-08-01

Riboswitches are a type of natural genetic control element that use untranslated sequence in the RNA to recognize and bind to small molecules that regulate expression of that gene. Creation of synthetic riboswitches to novel ligands depends on the ability to screen for analyte binding sensitivity and specificity. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically-coded fluorescent proteins. Specifically, a theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence, and a FRET-based construct, BFP-eGFP or eGFP-REACh, was linked by a peptide encoding the recognition sequence for TEV protease. Cells expressing the riboswitch showed a marked optical difference in fluorescence emission in the presence of theophylline. However, the BFP-eGFP FRET pair posses significant optical background that reduces the sensitivity of a FRET-based assay. To improve the optical assay, we designed a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein) as the FRET acceptor for eGFP. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence which leads to an improved detection of FRET via better signal-to-noise ratio. The EGFP-REACh fusion protein was constructed with the TEV protease cleavage site; thus upon TEV translation, cleavage occurs diminishing REACh quenching and increasing eGFP emission resulting in a 4.5-fold improvement in assay sensitivity.

Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA.

PubMed Central

Kirkegaard, K; Nelsen, B

1990-01-01

Small deletions were introduced into DNA plasmids bearing cDNA copies of Mahoney type 1 poliovirus RNA. The procedure used was similar to that of P. Hearing and T. Shenk (J. Mol. Biol. 167:809-822, 1983), with modifications designed to introduce only one lesion randomly into each DNA molecule. Methods to map small deletions in either large DNA or RNA molecules were employed. Two poliovirus mutants, VP1-101 and VP1-102, were selected from mutagenized populations on the basis of their host range phenotype, showing a large reduction in the relative numbers of plaques on CV1 and HeLa cells compared with wild-type virus. The deletions borne by the mutant genomes were mapped to the region encoding the amino terminus of VP1. That these lesions were responsible for the mutant phenotypes was substantiated by reintroduction of the sequenced lesions into a wild-type poliovirus cDNA by deoxyoligonucleotide-directed mutagenesis. The deletion of nucleotides encoding amino acids 8 and 9 of VP1 was responsible for the VP1-101 phenotype; the VP1-102 defect was caused by the deletion of the sequences encoding the first four amino acids of VP1. The peptide sequence at the VP1-VP3 proteolytic cleavage site was altered from glutamine-glycine to glutamine-methionine in VP1-102; this apparently did not alter the proteolytic cleavage pattern. The biochemical defects resulting from these mutations are discussed in the accompanying report. Images PMID:2152811

Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.

PubMed Central

Gilley, D; Preer, J R; Aufderheide, K J; Polisky, B

1988-01-01

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences. Images PMID:3211128

Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast *

PubMed Central

Clauser, Karl R.; Shen, Hongying; Kamer, Kimberli J.; Wells, James A.

2017-01-01

The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney. Comparative analysis between mitochondrial N-termini from mouse, human, and yeast proteins shows that whereas presequences are poorly conserved at the sequence level, other presequence properties are extremely conserved, including a length of ∼20–60 amino acids, a net charge between +3 to +6, and the presence of stabilizing amino acids at the N-terminus of mature proteins that follow the N-end rule from bacteria. As in yeast, ∼80% of mouse presequence cleavage sites match canonical motifs for three mitochondrial peptidases (MPP, Icp55, and Oct1), whereas the remainder do not match any known peptidase motifs. We show that mature mitochondrial proteins often exist with a spectrum of N-termini, consistent with a model of multiple cleavage events by MPP and Icp55. In addition to analysis of canonical targeting presequences, our N-terminal dataset allows the exploration of other cleavage events and provides support for polypeptide cleavage into two distinct enzymes (Hsd17b4), protein cleavages key for signaling (Oma1, Opa1, Htra2, Mavs, and Bcs2l13), and in several cases suggests novel protein isoforms (Scp2, Acadm, Adck3, Hsdl2, Dlst, and Ogdh). We present an integrated catalog of mammalian mitochondrial N-termini that can be used as a community resource to investigate individual proteins, to elucidate mechanisms of mammalian mitochondrial processing, and to allow researchers to engineer tags distally to the presequence cleavage. PMID:28122942

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry.

PubMed

Gardner, Qurra-tul-Ann Afza; Younas, Hooria; Akhtar, Muhammad

2013-01-01

Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k(cat.)/K(m) values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10(5)s(-1)M(-1)) and the cleavage of the K(29)-T(30) bond of M-insulin-RR (1.3±0.07×10(5)s(-1)M(-1)). However, the K(29)-T(30) bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k(cat.)/K(m) values around 1000s(-1)M(-1)). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K(29)-T(30) bond becomes shielded, exposed then shielded again respectively. Copyright © 2012 Elsevier B.V. All rights reserved.

An isoleucine to leucine mutation that switches the cofactor requirement of the EcoRV restriction endonuclease from magnesium to manganese.

PubMed

Vipond, I B; Moon, B J; Halford, S E

1996-02-13

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence more readily with Mg2+ as the cofactor than with Mn2+ but, at noncognate sequences that differ from the EcoRV site by one base pair, Mn2+ gives higher rates than Mg2+. A mutant of EcoRV, in which an isoleucine near the active site was replaced by leucine, showed the opposite behavior. It had low activity with Mg2+, but, in the presence of Mn2+ ions, it cleaved the recognition site faster than wild-type EcoRV with either Mn2+ or Mg2+. The mutant was also more specific for the recognition sequence than the native enzyme: the noncognate DNA cleavages by wild-type EcoRV and Mn2+ were not detected with the mutant. Further mutagenesis showed that the protein required the same acidic residues at its active site as wild-type EcoRV. The Ile-->Leu mutation seems to perturb the configuration of the metal-binding ligands at the active site so that the protein has virtually no affinity for Mg2+ yet it can still bind Mn2+ ions, though the latter only occurs when the protein is at the recognition site. This contrasts to wild-type EcoRV, where Mn2+ ions bind readily to complexes with either cognate and noncognate DNA and only Mg2+ shows the discrimination between the complexes. The structural perturbation is a specific consequence of leucine in place of isoleucine, since mutants with valine or alanine were similar to wild-type EcoRV.

cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage.

PubMed

Baron, Alice; Verdié, Pascal; Martinez, Jean; Lamaty, Frédéric

2011-02-04

A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.

Characterization of avian paramyxovirus serotype 14, a novel serotype, isolated from a duck fecal sample in Japan.

PubMed

Thampaisarn, Rapeewan; Bui, Vuong N; Trinh, Dai Q; Nagai, Makoto; Mizutani, Tetsuya; Omatsu, Tsutomu; Katayama, Yukie; Gronsang, Dulyatad; Le, Duong H T; Ogawa, Haruko; Imai, Kunitoshi

2017-01-15

A hemagglutinating virus isolate designated 11OG0352, was obtained from a duck fecal sample. Genetic and virological analyses indicated that it might represent a novel serotype of avian paramyxovirus (APMV). Electron micrographs showed that the morphology of the virus particle was similar to that of APMV. The complete genome of this virus comprised 15,444 nucleotides complying with the paramyxovirus "rule of six" and contains six open reading frames (3'-N-P-M-F-HN-L-5'). The phylogenetic analysis of the whole genome revealed that the virus was a member of the genus Avulavirus, but that it was distinct from APMV-1 to APMV-13. Although the F-protein cleavage site was TREGK↓L, which resembles a lentogenic strain of APMV-1, the K residue at position -1 of the cleavage site was first discovered in APMV members. The phosphoprotein gene of isolate 11OG0352 contains a putative RNA editing site, 3'-AUUUUCCC-5' (negative sense) which sequence differs from that of other APMVs. The intracerebral pathogenicity index test did not detect virulence in infected chicks. In hemagglutination inhibition (HI) tests, an antiserum against this virus did not detectably react with other APMVs (serotypes 1-4, 6-9) except for low reciprocal cross-reactivity with APMV-6. We designated this isolate, as APMV-14/duck/Japan/11OG0352/2011 and propose that it is a novel APMV serotype. The HI test may not be widely applicable for the classification of a new serotype because of the limited availability of reference antisera against all serotypes and cross-reactivity data. The nucleotide sequence identities of the whole genome of 11OG0352 and other APMVs ranged from 46.3% to 56.1%. Such comparison may provide a useful tool for classifying new APMV isolates. However, the nucleotide sequence identity between APMV-12 and APMV-13 was higher (64%), which was nearly identical to the lowest nucleotide identity (67%) reported in subgroups within the serotype. Therefore, consensus criteria for using whole genome analysis should be established. Copyright © 2016 Elsevier B.V. All rights reserved.

Specific Cleavage of the Nucleoprotein of Fish Rhabdovirus.

PubMed

Zhou, G-Z; Yi, Y-J; Chen, Z-Y; Zhang, Q-Y

2015-11-01

Siniperca chuatsi rhabdovirus (SCRV) is one of myriad rhabdoviruses recorded in fish. Preliminary data show that inhibition of the SCRV nucleoprotein (N) could significantly reduce the progeny virus titers in infected Epithelioma papulosum cyprinid (EPC) cells. Here, the authors propose that cleavage of the viral 47-kDa N protein is caspase-mediated based on caspase inhibition experiments, transient expression in EPC transfection, and analysis of cleavage sites. Cleavage of the SCRV N protein in culture was prevented by a pan-caspase inhibitor, z-VAD-FMK (z-Val-Ala-DL-Asp-fluoromethyl ketone). Subsequently, N was transiently expressed in EPC cells, the results of which indicated that the specific cleavage of N also occurred in the cells transfected with N-GFP plasmid. Several truncated fragments of the N gene were constructed and transiently transfected into EPC cells. Immunoblotting results indicated that D324 and D374 are the cleavage sites of N by caspases. The authors also found that z-VAD-FMK could inhibit the cytopathic effect in SCRV-infected EPC cells but not affect the production of infectious progeny, suggesting that the caspase-mediated cleavage of N protein is not required for in vitro SCRV replication. To the authors' knowledge, this is the first report on the cleavage of rhabdovirus proteins. © The Author(s) 2015.

Four new type I restriction enzymes identified in Escherichia coli clinical isolates

PubMed Central

Kasarjian, Julie K. A.; Kodama, Yoshiaki; Iida, Masatake; Matsuda, Katsura; Ryu, Junichi

2005-01-01

Using a plasmid transformation method and the RM search computer program, four type I restriction enzymes with new recognition sites and two isoschizomers (EcoBI and Eco377I) were identified in a collection of clinical Escherichia coli isolates. These new enzymes were designated Eco394I, Eco826I, Eco851I and Eco912I. Their recognition sequences were determined to be GAC(5N)RTAAY, GCA(6N)CTGA, GTCA(6N)TGAY and CAC(5N)TGGC, respectively. A methylation sensitivity assay, using various synthetic oligonucleotides, was used to identify the adenines that prevent cleavage when methylated (underlined). These results suggest that type I enzymes are abundant in E.coli and many other bacteria, as has been inferred from bacterial genome sequencing projects. PMID:16040596

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

PubMed

Mattijssen, Sandy; Hinson, Ella R; Onnekink, Carla; Hermanns, Pia; Zabel, Bernhard; Cresswell, Peter; Pruijn, Ger J M

2011-07-01

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

Structure of homeodomain-leucine zipper/DNA complexes studied using hydroxyl radical cleavage of DNA and methylation interference.

PubMed

Tron, Adriana E; Comelli, Raúl N; Gonzalez, Daniel H

2005-12-27

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.

'Cold shock' increases the frequency of homology directed repair gene editing in induced pluripotent stem cells.

PubMed

Guo, Q; Mintier, G; Ma-Edmonds, M; Storton, D; Wang, X; Xiao, X; Kienzle, B; Zhao, D; Feder, John N

2018-02-01

Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20-40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1-20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by 'cold shocking' cells at 32 °C for 24-48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or 'perfect HDR', as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated ('partial HDR'). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing 'perfect HDR' compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

PubMed

Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-15

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors. Copyright © 2017 American Society for Microbiology.

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity.

PubMed

Coppola, Julia M; Hamilton, Christin A; Bhojani, Mahaveer S; Larsen, Martha J; Ross, Brian D; Rehemtulla, Alnawaz

2007-05-01

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.

Searching for discrimination rules in protease proteolytic cleavage activity using genetic programming with a min-max scoring function.

PubMed

Yang, Zheng Rong; Thomson, Rebecca; Hodgman, T Charles; Dry, Jonathan; Doyle, Austin K; Narayanan, Ajit; Wu, XiKun

2003-11-01

This paper presents an algorithm which is able to extract discriminant rules from oligopeptides for protease proteolytic cleavage activity prediction. The algorithm is developed using genetic programming. Three important components in the algorithm are a min-max scoring function, the reverse Polish notation (RPN) and the use of minimum description length. The min-max scoring function is developed using amino acid similarity matrices for measuring the similarity between an oligopeptide and a rule, which is a complex algebraic equation of amino acids rather than a simple pattern sequence. The Fisher ratio is then calculated on the scoring values using the class label associated with the oligopeptides. The discriminant ability of each rule can therefore be evaluated. The use of RPN makes the evolutionary operations simpler and therefore reduces the computational cost. To prevent overfitting, the concept of minimum description length is used to penalize over-complicated rules. A fitness function is therefore composed of the Fisher ratio and the use of minimum description length for an efficient evolutionary process. In the application to four protease datasets (Trypsin, Factor Xa, Hepatitis C Virus and HIV protease cleavage site prediction), our algorithm is superior to C5, a conventional method for deriving decision trees.

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome

PubMed Central

ORBAN, TAMAS I.; IZAURRALDE, ELISA

2005-01-01

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21–22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5′ mRNA fragments generated by RISC cleavage are rapidly degraded from their 3′ ends by the exosome, whereas the 3′ fragments are degraded from their 5′ ends by XRN1. Exosome-mediated decay of the 5′ fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation. PMID:15703439

Identification of inhibitors using a cell based assay for monitoring golgi-resident protease activity

PubMed Central

Coppola, Julia M.; Hamilton, Christin A.; Bhojani, Mahaveer S.; Larsen, Martha J.; Ross, Brian D.; Rehemtulla, Alnawaz

2007-01-01

Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE. PMID:17316541

Crystal Structures of Yellowtail Ascites Virus VP4 Protease

PubMed Central

Chung, Ivy Yeuk Wah; Paetzel, Mark

2013-01-01

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala716) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser633 as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water. PMID:23511637

Site-specific Phosphorylation Protects Glycogen Synthase Kinase-3β from Calpain-mediated Truncation of Its N and C Termini*

PubMed Central

Ma, Shanshan; Liu, Shaojun; Huang, Qiaoying; Xie, Bo; Lai, Bingquan; Wang, Chong; Song, Bin; Li, Mingtao

2012-01-01

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38–Thr-39 and Ile-384–Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39–420), ΔC-GSK-3β (amino acids 1–384), and ΔN/ΔC-GSK-3β (amino acids 39–384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity. PMID:22496446

Site-specific phosphorylation protects glycogen synthase kinase-3β from calpain-mediated truncation of its N and C termini.

PubMed

Ma, Shanshan; Liu, Shaojun; Huang, Qiaoying; Xie, Bo; Lai, Bingquan; Wang, Chong; Song, Bin; Li, Mingtao

2012-06-29

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38-Thr-39 and Ile-384-Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39-420), ΔC-GSK-3β (amino acids 1-384), and ΔN/ΔC-GSK-3β (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity.

Anti-amyloid precursor protein antibodies inhibit amyloid-β production by steric hindrance

PubMed Central

Thomas, Rhian S.; Liddell, J. Eryl; Kidd, Emma J.

2015-01-01

Summary Cleavage of amyloid precursor protein (APP) by β- and γ-secretases results in the production of amyloid-β (Aβ) in Alzheimer’s disease (AD). We raised two monoclonal antibodies, 2B3 and 2B12, that recognise the β-secretase cleavage site on APP but not Aβ. We hypothesised that these antibodies would reduce Aβ levels via steric hindrance of β-secretase. Both antibodies decreased extracellular Aβ levels from astrocytoma cells, but 2B3 was more potent than 2B12. Levels of soluble sAPPα from the non-amyloidogenic α-secretase pathway and intracellular APP were not affected by either antibody nor were there any effects on cell viability. 2B3 exhibited a higher affinity for APP than 2B12 and its epitope appeared to span the cleavage site while 2B12 bound slightly upstream. Both of these factors probably contribute to its greater effect on Aβ levels. After 60 minutes incubation at pH 4.0, most 2B3 and 2B12 remained bound to their antigen, suggesting that the antibodies will remain bound to APP in the acidic endosomes where β-secretase cleavage probably occurs. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPPα by β-secretase in a cell-free assay where effects of antibody internalisation and intracellular degradation were excluded. 2B3 virtually abolished this cleavage. In addition, levels of C-terminal APP fragments, βCTF, generated following β-secretase cleavage, were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site antibodies can generically reduce Aβ levels via inhibition of β-secretase by steric hindrance and may provide a novel alternative therapy for AD. PMID:21122073

Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903.

PubMed Central

Grindley, N D; Joyce, C M

1980-01-01

The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences. Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs. We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats. Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient. One such transposable derivative, Tn903 delta I, has been selected for further study. We have determined the sequence of the intact inverted repeat. The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences. Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903. To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability. The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903. Images PMID:6261245

The C-terminal portion of the cleaved HT motif is necessary and sufficient to mediate export of proteins from the malaria parasite into its host cell

PubMed Central

Tarr, Sarah J; Cryar, Adam; Thalassinos, Konstantinos; Haldar, Kasturi; Osborne, Andrew R

2013-01-01

The malaria parasite exports proteins across its plasma membrane and a surrounding parasitophorous vacuole membrane, into its host erythrocyte. Most exported proteins contain a Host Targeting motif (HT motif) that targets them for export. In the parasite secretory pathway, the HT motif is cleaved by the protease plasmepsin V, but the role of the newly generated N-terminal sequence in protein export is unclear. Using a model protein that is cleaved by an exogenous viral protease, we show that the new N-terminal sequence, normally generated by plasmepsin V cleavage, is sufficient to target a protein for export, and that cleavage by plasmepsin V is not coupled directly to the transfer of a protein to the next component in the export pathway. Mutation of the fourth and fifth positions of the HT motif, as well as amino acids further downstream, block or affect the efficiency of protein export indicating that this region is necessary for efficient export. We also show that the fifth position of the HT motif is important for plasmepsin V cleavage. Our results indicate that plasmepsin V cleavage is required to generate a new N-terminal sequence that is necessary and sufficient to mediate protein export by the malaria parasite. PMID:23279267

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA.

PubMed

Horton, John R; Borgaro, Janine G; Griggs, Rose M; Quimby, Aine; Guan, Shengxi; Zhang, Xing; Wilson, Geoffrey G; Zheng, Yu; Zhu, Zhenyu; Cheng, Xiaodong

2014-07-01

AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ∼70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ∼22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horton, John R.; Borgaro, Janine G.; Griggs, Rose M.

2014-07-03

AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves DNA containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises anmore » N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ~ 70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ~ 22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.« less

Conformational changes in the P site and mRNA entry channel evoked by AUG recognition in yeast translation preinitiation complexes

PubMed Central

Zhang, Fan; Saini, Adesh K.; Shin, Byung-Sik; Nanda, Jagpreet; Hinnebusch, Alan G.

2015-01-01

The translation preinitiation complex (PIC) is thought to assume an open conformation when scanning the mRNA leader, with AUG recognition evoking a closed conformation and more stable P site interaction of Met-tRNAi; however, physical evidence is lacking that AUG recognition constrains interaction of mRNA with the 40S binding cleft. We compared patterns of hydroxyl radical cleavage of rRNA by Fe(II)-BABE tethered to unique sites in eIF1A in yeast PICs reconstituted with mRNA harboring an AUG or near-cognate (AUC) start codon. rRNA residues in the P site display reduced cleavage in AUG versus AUC PICs; and enhanced cleavage in the AUC complexes was diminished by mutations of scanning enhancer elements of eIF1A that increase near-cognate recognition in vivo. This suggests that accessibility of these rRNA residues is reduced by accommodation of Met-tRNAi in the P site (PIN state) and by their interactions with the anticodon stem of Met-tRNAi. Our cleavage data also provide evidence that AUG recognition evokes dissociation of eIF1 from its 40S binding site, ejection of the eIF1A-CTT from the P-site and rearrangement to a closed conformation of the entry channel with reduced mobility of mRNA. PMID:25670678

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by methidiumpropyl.EDTA and localization of the major dye binding sites.

PubMed Central

Tanner, N K; Cech, T R

1985-01-01

The intervening sequence (IVS) excised from the rRNA precursor of Tetrahymena thermophila is converted to a covalently closed circular RNA in the absence of proteins in vitro. This self-catalyzed cyclization reaction is inhibited by the intercalating dye methidiumpropyl.EDTA (MPE; R.P. Hertzberg and P.B. Dervan (1982) J. Am. Chem. Soc. 104, 313-315). The MPE binding sites have been localized by mapping the sites of MPE.Fe(II) cleavage of the IVS RNA. There are three major binding sites within the 414 nucleotide IVS RNA. Two of these sites coincide with the A.B and 9L.2 pairings. These are structural elements that are conserved in all group I introns and are implicated as being functionally important for splicing. We propose that interaction of MPE with these sites is responsible for dye inhibition of cyclization. The reactions of MPE.Fe(II) with an RNA of known structure, tRNAPhe, and with the IVS RNA were studied as a function of temperature, ionic strength and ethidium concentration. Based on the comparison of the reaction with these two RNAs, we conclude that the dye is a very useful probe for structural regions of large RNAs, while it provides more limited structural information about the small, compact tRNA molecule. Images PMID:2415924

The complete genome sequence of freesia mosaic virus and its relationship to other potyviruses.

PubMed

Choi, H I; Lim, H R; Song, Y S; Kim, M J; Choi, S H; Song, Y S; Bae, S C; Ryu, K H

2010-07-01

We have completed the genomic sequence of a potyvirus, freesia mosaic virus (FreMV), and compared it to those of other known potyviruses. The full-length genome sequence of FreMV consists of 9,489 nucleotides. The large protein contains 3,077 amino acids, with an AUG start codon and UAA stop codon, containing one open reading frame typical of a potyvirus polyprotein. The polyprotein of FreMV-Kr gives rise to eleven proteins (P1, HC-pro, P3, PIPO, 6K1, CI, 6K2, VPg, NIa, NIb and CP), and putative cleavage sites of each protein were identified by sequence comparison to those of other known potyviruses. Phylogenetic analysis of the polyprotein revealed that FreMV-Kr was most closely related to PeMoV and was related to BtMV, BaRMV and PeLMV, which belong to the BCMV subgroup. This is the first information on the complete genome structure of FreMV, and the sequence information clearly supports the status of FreMV as a member of a distinct species in the genus Potyvirus.

Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ɛ-cleavage site

NASA Astrophysics Data System (ADS)

Chen, Wen; Gamache, Eric; Rosenman, David J.; Xie, Jian; Lopez, Maria M.; Li, Yue-Ming; Wang, Chunyu

2014-01-01

The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer’s disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ɛ-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ɛ-cleavage, and consequently shift cleavage preference towards Aβ42.

Classical Swine Fever Virus Glycoprotein Erns Is an Endoribonuclease with an Unusual Base Specificity

PubMed Central

Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till

2004-01-01

The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930

Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

PubMed Central

Subramanian, Shoba; Hardt, Markus; Choe, Youngchool; Niles, Richard K.; Johansen, Eric B.; Legac, Jennifer; Gut, Jiri; Kerr, Iain D.; Craik, Charles S.; Rosenthal, Philip J.

2009-01-01

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P1 – P4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P2 position. Second, with overlapping peptides spanning α and β globin and proteolysis-dependent 18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents. PMID:19357776

Avian paramyxovirus serotype 1 strains of low virulence with unusual fusion protein cleavage sites isolated from poultry species

USDA-ARS?s Scientific Manuscript database

Avian paramyxo-serotype-1 viruses (APMV1) with fusion cleavage sites containing two basic amino acids and a phenylalanine (F) at position 117 have been isolated from poultry species in two states from 2007-2009. The intracerebral pathogenicity indices for these viruses are of low virulence at 0.00 ...

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases

PubMed Central

Lambert, Abigail R.; Kuhar, Ryan; Jarjour, Jordan; Kulshina, Nadia; Parmeggiani, Fabio; Danaher, Patrick; Gano, Jacob; Baker, David; Stoddard, Barry L.; Scharenberg, Andrew M.

2012-01-01

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40–70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ∼70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications. PMID:22684507

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

PubMed

Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

2015-10-09

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Photoelectrochemical Biosensor for Tyrosinase and Thrombin Detection

PubMed Central

Chen, Jiexia; Liu, Yifan; Zhao, Guang-Chao

2016-01-01

A novel photoelectrochemical biosensor for step-by-step assay of tyrosinase and thrombin was fabricated based on the specific interactions between the designed peptide and the target enzymes. A peptide chain with a special sequence which contains a positively charged lysine-labeled terminal, tyrosine at the other end and a cleavage site recognized by thrombin between them was designed. The designed peptide can be fixed on surface of the CdTe quantum dots (QDs)-modified indium-tin oxide (ITO) electrode through electrostatic attraction to construct the photoelectrochemical biosensor. The tyrosinase target can catalyze the oxidization of tyrosine by oxygen into ortho-benzoquinone residues, which results in a decrease in the sensor photocurrent. Subsequently, the cleavage site could be recognized and cut off by another thrombin target, restoring the sensor photocurrent. The decrease or increase of photocurrent in the sensor enables us to assay tyrosinase and thrombin. Thus, the detection of tyrosinase and thrombin can be achieved in the linear range from 2.6 to 32 μg/mL and from 4.5 to 100 μg/mL with detection limits of 1.5 μg/mL and 1.9 μg/mL, respectively. Most importantly, this strategy shall allow us to detect different classes of enzymes simultaneously by designing various enzyme-specific peptide substrates. PMID:26805846

The Evolutionary Pattern of Glycosylation Sites in Influenza Virus (H5N1) Hemagglutinin and Neuraminidase

PubMed Central

Chen, Wentian; Zhong, Yaogang; Qin, Yannan; Sun, Shisheng; Li, Zheng

2012-01-01

Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of influenza viruses play crucial roles in transfaunation, membrane fusion and the release of progeny virions. To explore the distribution of N-glycosylation sites (glycosites) in these two glycoproteins, we collected and aligned the amino acid sequences of all the HA and NA subtypes. Two glycosites were located at HA0 cleavage sites and fusion peptides and were strikingly conserved in all HA subtypes, while the remaining glycosites were unique to their subtypes. Two to four conserved glycosites were found in the stalk domain of NA, but these are affected by the deletion of specific stalk domain sequences. Another highly conserved glycosite appeared at the top center of tetrameric global domain, while the others glycosites were distributed around the global domain. Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, and further focus on the H5N1 virus and conclude that the glycosites in H5N1 have become more complicated in HA and less influential in NA in the last five years. PMID:23133677

Cleavage reaction of HDV ribozymes in the presence of Mg2+ is accompanied by a conformational change.

PubMed

Tanaka, Yoichiro; Tagaya, Mitsuhiro; Hori, Tamaki; Sakamoto, Taiichi; Kurihara, Yasuyuki; Katahira, Masato; Uesugi, Seiichi

2002-06-01

Hepatitis delta virus (HDV) ribozymes cleave RNA in the presence of divalent metal ions. We have previously elucidated the solution conformation of a minimized trans-acting HDV ribozyme and obtained evidence by NMR study that an Mg2+ ion binds to a site close to the cleavage site. We examined two ribozyme systems: a pre-cleavage complex with a non-cleavable substrate analogue (mS8) and a post-cleavage complex with a 3' cleavage product (P7). Upon titration with MgCl2, the complex with P7 showed a profound spectral change, while that with mS8 showed broadening of the signals. Analysis of the NOESY spectra of the P7 complex at high Mg2+ concentration revealed that a G:U pair is formed within the L3 loop, and the P1 and P4 stems are stabilized with respect to those of the pre-cleavage complex. The present analysis indicates that the cleavage reaction of the HDV ribozyme produces a big conformational change. Furthermore, presence of the 5'-terminal cytidine residue prevents this conformational change and its absence stabilizes the product-ribozyme complex in the presence of Mg2+. The structure of the Mg2+-bound P7 complex is similar to the crystal structure found for a product-ribozyme complex but is different from the pre-cleavage structure.

Converting Transaldolase into Aldolase through Swapping of the Multifunctional Acid-Base Catalyst: Common and Divergent Catalytic Principles in F6P Aldolase and Transaldolase.

PubMed

Sautner, Viktor; Friedrich, Mascha Miriam; Lehwess-Litzmann, Anja; Tittmann, Kai

2015-07-28

Transaldolase (TAL) and fructose-6-phosphate aldolase (FSA) both belong to the class I aldolase family and share a high degree of structural similarity and sequence identity. The molecular basis of the different reaction specificities (transferase vs aldolase) has remained enigmatic. A notable difference between the active sites is the presence of either a TAL-specific Glu (Gln in FSA) or a FSA-specific Tyr (Phe in TAL). Both residues seem to have analoguous multifunctional catalytic roles but are positioned at different faces of the substrate locale. We have engineered a TAL double variant (Glu to Gln and Phe to Tyr) with an active site resembling that of FSA. This variant indeed exhibits aldolase activity as its main activity with a catalytic efficiency even larger than that of authentic FSA, while TAL activity is greatly impaired. Structural analysis of this variant in complex with the dihydroxyacetone Schiff base formed upon substrate cleavage identifies the introduced Tyr (genuine in FSA) to catalyze protonation of the central carbanion-enamine intermediate as a key determinant of the aldolase reaction. Our studies pinpoint that the Glu in TAL and the Tyr in FSA, although located at different positions at the active site, similarly act as bona fide acid-base catalysts in numerous catalytic steps, including substrate binding, dehydration of the carbinolamine, and substrate cleavage. We propose that the different spatial positions of the multifunctional Glu in TAL and of the corresponding multifunctional Tyr in FSA relative to the substrate locale are critically controlling reaction specificity through either unfavorable (TAL) or favorable (FSA) geometry of proton transfer onto the common carbanion-enamine intermediate. The presence of both potential acid-base residues, Glu and Tyr, in the active site of TAL has deleterious effects on substrate binding and cleavage, most likely resulting from a differently organized H-bonding network. Large-scale motions of the protein associated with opening and closing of the active site that seem to bear relevance for catalysis are observed as covalent intermediates are exclusively observed in the "closed" conformation of the active site. Pre-steady-state kinetics are used to monitor catalytic processes and structural transitions and to refine the kinetic framework of TAL catalysis.

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly.

PubMed

VanBlargan, Laura A; Davis, Kaitlin A; Dowd, Kimberly A; Akey, David L; Smith, Janet L; Pierson, Theodore C

2015-08-01

The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1' position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1' position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1' position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause considerable morbidity and mortality in humans. No specific antiflavivirus therapeutics are available for treatment of infection. Proteolytic processing of the flavivirus polyprotein is an essential step in the replication cycle and is an attractive target for antiviral development. The design of protease inhibitors has been informed by insights into the molecular details of the interactions of proteases and their substrates. In this article, studies of the processing of WNV and DENV capsid proteins by the WNV protease identified an unexpected contribution of the sequence surrounding critical residues within the cleavage site on protease specificity. This demonstration of context-dependent protease cleavage has implications for the design of chimeric flaviviruses, new therapeutics, and the interpretation of flavivirus protease substrate specificity studies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

PubMed Central

VanBlargan, Laura A.; Davis, Kaitlin A.; Dowd, Kimberly A.; Akey, David L.; Smith, Janet L.

2015-01-01

ABSTRACT The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1′ position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1′ position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1′ position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. IMPORTANCE West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause considerable morbidity and mortality in humans. No specific antiflavivirus therapeutics are available for treatment of infection. Proteolytic processing of the flavivirus polyprotein is an essential step in the replication cycle and is an attractive target for antiviral development. The design of protease inhibitors has been informed by insights into the molecular details of the interactions of proteases and their substrates. In this article, studies of the processing of WNV and DENV capsid proteins by the WNV protease identified an unexpected contribution of the sequence surrounding critical residues within the cleavage site on protease specificity. This demonstration of context-dependent protease cleavage has implications for the design of chimeric flaviviruses, new therapeutics, and the interpretation of flavivirus protease substrate specificity studies. PMID:26063422

Cnidarian microRNAs frequently regulate targets by cleavage.

PubMed

Moran, Yehu; Fredman, David; Praher, Daniela; Li, Xin Z; Wee, Liang Meng; Rentzsch, Fabian; Zamore, Phillip D; Technau, Ulrich; Seitz, Hervé

2014-04-01

In bilaterians, which comprise most of extant animals, microRNAs (miRNAs) regulate the majority of messenger RNAs (mRNAs) via base-pairing of a short sequence (the miRNA "seed") to the target, subsequently promoting translational inhibition and transcript instability. In plants, many miRNAs guide endonucleolytic cleavage of highly complementary targets. Because little is known about miRNA function in nonbilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovers scores of novel miRNAs in Nematostella, increasing the total miRNA gene count to 87. Yet only a handful are conserved in corals and hydras, suggesting that microRNA gene turnover in Cnidaria greatly exceeds that of other metazoan groups. We further show that Nematostella miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity. This mode of action resembles that of small interfering RNAs (siRNAs) and plant miRNAs. It appears to be common in Cnidaria, as several of the miRNA target sites are conserved among distantly related anemone species, and we also detected miRNA-directed cleavage in Hydra. Unlike in bilaterians, Nematostella miRNAs are commonly coexpressed with their target transcripts. In light of these findings, we propose that post-transcriptional regulation by miRNAs functions differently in Cnidaria and Bilateria. The similar, siRNA-like mode of action of miRNAs in Cnidaria and plants suggests that this may be an ancestral state.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas

Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 thatmore » forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.« less

The action of neutrophil serine proteases on elastin and its precursor.

PubMed

Heinz, Andrea; Jung, Michael C; Jahreis, Günther; Rusciani, Anthony; Duca, Laurent; Debelle, Laurent; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

2012-01-01

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P(1) in tropoelastin, whereas Lys was not identified at P(1) in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P(2) and P(4)'. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

A Novel Activation Mechanism of Avian Influenza Virus H9N2 by Furin

PubMed Central

Tse, Longping V.; Hamilton, Alice M.; Friling, Tamar

2014-01-01

Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA1 allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses. PMID:24257604

An asparagine residue at the N-terminus affects the maturation process of low molecular weight glutenin subunits of wheat endosperm

PubMed Central

2014-01-01

Background Wheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain. It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase. Results We performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene. Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing. Conclusions Our results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence. PMID:24629124

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

PubMed

Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander; Adey, Andrew; Gouble, Agnès; Duchateau, Philippe; Shendure, Jay; Stoddard, Barry L; Certo, Michael T; Baker, David; Scharenberg, Andrew M

2014-02-01

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

A nucleotide sequence comparison of coxsackievirus B4 isolates from aquatic samples and clinical specimens.

PubMed Central

Hughes, M. S.; Hoey, E. M.; Coyle, P. V.

1993-01-01

Ten coxsackievirus B4 (CVB4) strains isolated from clinical and environmental sources in Northern Ireland in 1985-7, were compared at the nucleotide sequence level. Dideoxynucleotide sequencing of a polymerase chain reaction (PCR) amplified fragment, spanning the VP1/P2A genomic region, classified the isolates into two distinct groups or genotypes as defined by Rico-Hesse and colleagues for poliovirus type 1. Isolates within each group shared approximately 99% sequence identity at the nucleotide level whereas < or = 86% sequence identity was shared between groups. One isolate derived from a clinical specimen in 1987 was grouped with six CVB4 isolates recovered from the aquatic environment in 1986-7. The second group comprised CVB4 isolates from clinical specimens in 1985-6. Both groups were different at the nucleotide level from the prototype strain isolated in 1950. It was concluded that the method could be used to sub-type CVB4 isolates and would be of value in epidemiological studies of CVB4. Predicted amino acid sequences revealed non-conservation of the tyrosine residue at the VP1/P2A cleavage site but were of little value in distinguishing CVB4 variants. PMID:8386098

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartley, J.A.; Forrow, S.M.; Souhami, R.L.

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increase ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specificmore » chemical cleavage technique for DNA sequencing. The result differed with both the nitrogen mustard and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.« less

Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

PubMed

Han, Yao; Zhang, Bin; Qin, Xiaoting; Li, Mingyang; Guo, Yulong

2015-01-01

MIGS (miRNA-induced gene silencing) is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs). MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase) and PDS (phytoene desaturase) gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant functions.

Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes

PubMed Central

Han, Yao; Zhang, Bin; Qin, Xiaoting; Li, Mingyang; Guo, Yulong

2015-01-01

MIGS (miRNA-induced gene silencing) is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs). MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase) and PDS (phytoene desaturase) gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5′- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant functions. PMID:26658695

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus.

PubMed

Qiu, T; Lu, R H; Zhang, J; Zhu, Z Y

2001-07-01

The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mu1 of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mu1.

Metagenome Sequence Analysis of Filamentous Microbial Communities Obtained from Geochemically Distinct Geothermal Channels Reveals Specialization of Three Aquificales Lineages

PubMed Central

Takacs-Vesbach, Cristina; Inskeep, William P.; Jay, Zackary J.; Herrgard, Markus J.; Rusch, Douglas B.; Tringe, Susannah G.; Kozubal, Mark A.; Hamamura, Natsuko; Macur, Richard E.; Fouke, Bruce W.; Reysenbach, Anna-Louise; McDermott, Timothy R.; Jennings, Ryan deM.; Hengartner, Nicolas W.; Xie, Gary

2013-01-01

The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal “filamentous streamer” communities (∼40 Mbp per site), which targeted three different groups of Aquificales found in Yellowstone National Park (YNP). Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobaculum (Aquificaceae) populations, whereas the circum-neutral pH (6.5–7.8) sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae). Thermocrinis (Aquificaceae) populations were found primarily in the circum-neutral sites with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phylogenetic analysis of assembled sequence containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse-TCA cycle, but only the Sulfurihydrogenibium populations perform citrate cleavage using ATP citrate lyase (Acl). The Aquificaceae populations use an alternative pathway catalyzed by two separate enzymes, citryl-CoA synthetase (Ccs), and citryl-CoA lyase (Ccl). All three Aquificales lineages contained evidence of aerobic respiration, albeit due to completely different types of heme Cu oxidases (subunit I) involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2) have resulted in niche specialization among members of the Aquificales. PMID:23755042

Characterization of three peptides derived from prosomatostatin [prosomatostatin-(1-63)-, -(65-76)- and -(79-92)-peptides] in a human pancreatic tumour.

PubMed

Conlon, J M; Eriksson, B; Grimelius, L; Oberg, K; Thim, L

1987-11-15

By using only reverse-phase h.p.l.c., three fragments of prosomatostatin were isolated from an extract of a human pancreatic neuroendocrine tumour that produced somatostatin, vasoactive intestinal polypeptide and gastrin-releasing peptide. The amino acid composition of the peptides indicated that they represented prosomatostatin-(1-63)-peptide, prosomatostain-(65-76)-peptide and prosomatostatin-(79-92)-peptide (somatostatin-14). The identity of prosomatostatin-(1-63)-peptide was confirmed by characterization of the products of digestion with Armillaria mellea (honey fungus) proteinase. Partial micro-sequencing of prosomatostatin-(1-63)-peptide showed that the Gly24-Ala25 bond of preprosomatostatin was the site of cleavage of the signal peptide. Thus human prosomatostatin is a protein of 92 amino acid residues that is proteolytically cleaved in a pancreatic tumour at the site of a dibasic-residue (arginine-lysine) processing site and at a single-monobasic-residue (arginine) processing site.

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Masahito; Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571; Umeyama, Kazuhiro

2010-11-05

Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor themore » exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.« less

Pre-lithification tectonic foliation development in a clastic sedimentary sequence

NASA Astrophysics Data System (ADS)

Meere, Patrick; Mulchrone, Kieran; McCarthy, David; Timmermann, Martin; Dewey, John

2016-04-01

The current view regarding the timing of regionally developed penetrative tectonic fabrics in sedimentary rocks is that their development postdates lithification of those rocks. In this case fabric development is achieved by a number of deformation mechanisms including grain rigid body rotation, crystal-plastic deformation and pressure solution (wet diffusion). The latter is believed to be the primary mechanism responsible for shortening and the domainal structure of cleavage development commonly observed in low grade metamorphic rocks. In this study we combine field observations with strain analysis and modelling to fully characterise considerable (>50%) mid-Devonian Acadian crustal shortening in a Devonian clastic sedimentary sequence from south west Ireland. Despite these high levels of shortening and associated penetrative tectonic fabric there is a marked absence of the expected domainal cleavage structure and intra-clast deformation, which are expected with this level of deformation. In contrast to the expected deformation processes associated with conventional cleavage development, fabrics in these rocks are a product of translation, rigid body rotation and repacking of extra-formational clasts during deformation of an un-lithified clastic sedimentary sequence.

[Construction and expression of a fusion protein made of tissue-type plasminogen activator and hirudin in Pichia pastoris].

PubMed

Yu, Ai-Ping; Shi, Bing-Xing; Dong, Chun-Na; Jiang, Zhong-Hua; Wu, Zu-Ze

2005-07-01

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.

Click chemistry-mediated cyclic cleavage of metal ion-dependent DNAzymes for amplified and colorimetric detection of human serum copper (II).

PubMed

Li, Daxiu; Xie, Jiaqing; Zhou, Wenjiao; Jiang, Bingying; Yuan, Ruo; Xiang, Yun

2017-11-01

The determination of the level of Cu 2+ plays important roles in disease diagnosis and environmental monitoring. By coupling Cu + -catalyzed click chemistry and metal ion-dependent DNAzyme cyclic amplification, we have developed a convenient and sensitive colorimetric sensing method for the detection of Cu 2+ in human serums. The target Cu 2+ can be reduced by ascorbate to form Cu + , which catalyzes the azide-alkyne cycloaddition between the azide- and alkyne-modified DNAs to form Mg 2+ -dependent DNAzymes. Subsequently, the Mg 2+ ions catalyze the cleavage of the hairpin DNA substrate sequences of the DNAzymes and trigger cyclic generation of a large number of free G-quadruplex sequences, which bind hemin to form the G-quadruplex/hemin artificial peroxidase to cause significant color transition of the sensing solution for sensitive colorimetric detection of Cu 2+ . This method shows a dynamic range of 5 to 500 nM and a detection limit of 2 nM for Cu 2+ detection. Besides, the level of Cu 2+ in human serums can also be determined by using this sensing approach. With the advantages of simplicity and high sensitivity, such sensing method thus holds great potential for on-site determination of Cu 2+ in different samples. Graphical abstract Sensitive colorimetric detection of copper (II) by coupling click chemistry with metal ion-dependentDNAzymes.

Computer-based prediction of mitochondria-targeting peptides.

PubMed

Martelli, Pier Luigi; Savojardo, Castrense; Fariselli, Piero; Tasco, Gianluca; Casadio, Rita

2015-01-01

Computational methods are invaluable when protein sequences, directly derived from genomic data, need functional and structural annotation. Subcellular localization is a feature necessary for understanding the protein role and the compartment where the mature protein is active and very difficult to characterize experimentally. Mitochondrial proteins encoded on the cytosolic ribosomes carry specific patterns in the precursor sequence from where it is possible to recognize a peptide targeting the protein to its final destination. Here we discuss to which extent it is feasible to develop computational methods for detecting mitochondrial targeting peptides in the precursor sequences and benchmark our and other methods on the human mitochondrial proteins endowed with experimentally characterized targeting peptides. Furthermore, we illustrate our newly implemented web server and its usage on the whole human proteome in order to infer mitochondrial targeting peptides, their cleavage sites, and whether the targeting peptide regions contain or not arginine-rich recurrent motifs. By this, we add some other 2,800 human proteins to the 124 ones already experimentally annotated with a mitochondrial targeting peptide.

CAPA-peptides of praying mantids (Mantodea).

PubMed

Koehler, Rene; Predel, Reinhard

2010-03-01

Dictyoptera which consist of cockroaches, termites, and praying mantids are among the oldest pterygote insects known. Whereas the localization and sequences of neuropeptides from a number of cockroaches are very well known, nearly nothing is known about the neuropeptides typical of praying mantids. In this study, the neuroanatomy of the median neuroendocrine system in the abdominal ventral nerve cord and the sequences of the CAPA-peptides which are expressed in the respective neuroendocrine cells were analyzed. Altogether, 40 species belonging to different families of Mantodea were included. In contrast to cockroaches, the mantids mostly express two CAPA-periviscerokinins (PVKs), only in Mantis religiosa a third PVK was identified. These PVKs are sequence-related to the PVKs of basal cockroaches (Polyphagidae). In a group of closely related Mantodea (Paramantinae), extended forms of PVK-2 were observed. As shown, these forms are possibly the result of substitutions in the N-terminal cleavage sites of the respective PVKs. No trace of a CAPA-pyrokinin was found in any of the praying mantids. (c) 2009 Elsevier Inc. All rights reserved.

In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus.

PubMed

Watanabe, Tsunamasa; Hatakeyama, Hiroto; Matsuda-Yasui, Chiho; Sato, Yusuke; Sudoh, Masayuki; Takagi, Asako; Hirata, Yuichi; Ohtsuki, Takahiro; Arai, Masaaki; Inoue, Kazuaki; Harashima, Hideyoshi; Kohara, Michinori

2014-04-23

The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.

Characterization and Epidemiology of Pigeon Paramyxovirus Type-1 Viruses (PPMV-1) Isolated in Macedonia.

PubMed

Dodovski, A; Cvetkovikj, I; Krstevski, K; Naletoski, I; Savić, Vladimir

2017-06-01

We have characterized in this study 10 PPMV-1 isolated from domestic pigeons and one PPMV-1 isolated from a feral pigeon in the period 2007-2012, using both classical methods (HI test and ICPI test) and molecular methods (RT-qPCR, RT-PCR, and nucleotide sequencing). Using phylogenetic analysis of partial fusion gene sequences, these viruses clustered with recent European PPMV-1 isolates (EU/re) within the genotype VIb/1. All isolates possessed virulent cleavage site motifs with variable morbidity and mortality in pigeons. The intracerebral pathogenecity indices of the five isolates ranged from 0.59 to 1.53. The repetitive isolation of PPMV-1 viruses for several consecutive years led toward establishing enzootic presence of the disease in pigeons. A high nucleotide sequence homology between the Macedonian isolates and EU/re isolates was shown. Co-circulation of different isolates in the same holdings was detected. This is the first study to extensively describe the molecular epidemiology of PPMV-1 isolated in Macedonia.

A bacterial Argonaute with noncanonical guide RNA specificity

PubMed Central

Kaya, Emine; Doxzen, Kevin W.; Knoll, Kilian R.; Wilson, Ross C.; Strutt, Steven C.; Kranzusch, Philip J.; Doudna, Jennifer A.

2016-01-01

Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-hydroxylated guide RNAs rather than the 5′-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo–RNA complex reveals a guide strand binding site comprising residues that block 5′ phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPR-cas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide. PMID:27035975

The Origins of Specificity in the Microcin-Processing Protease TldD/E.

PubMed

Ghilarov, Dmitry; Serebryakova, Marina; Stevenson, Clare E M; Hearnshaw, Stephen J; Volkov, Dmitry S; Maxwell, Anthony; Lawson, David M; Severinov, Konstantin

2017-10-03

TldD and TldE proteins are involved in the biosynthesis of microcin B17 (MccB17), an Escherichia coli thiazole/oxazole-modified peptide toxin targeting DNA gyrase. Using a combination of biochemical and crystallographic methods we show that E. coli TldD and TldE interact to form a heterodimeric metalloprotease. TldD/E cleaves the N-terminal leader sequence from the modified MccB17 precursor peptide, to yield mature antibiotic, while it has no effect on the unmodified peptide. Both proteins are essential for the activity; however, only the TldD subunit forms a novel metal-containing active site within the hollow core of the heterodimer. Peptide substrates are bound in a sequence-independent manner through β sheet interactions with TldD and are likely cleaved via a thermolysin-type mechanism. We suggest that TldD/E acts as a "molecular pencil sharpener": unfolded polypeptides are fed through a narrow channel into the active site and processively truncated through the cleavage of short peptides from the N-terminal end. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The effects of metal ions on the DNA damage induced by hydrogen peroxide.

PubMed

Kobayashi, S; Ueda, K; Komano, T

1990-01-01

The effects of metal ions on DNA damage induced by hydrogen peroxide were investigated using two methods, agarose-gel electrophoretic analysis of supercoiled DNA and sequencing-gel analysis of single end-labeled DNA fragments of defined sequences. Hydrogen peroxide induced DNA damage when iron or copper ion was present. At least two classes of DNA damage were induced, one being direct DNA-strand cleavage, and the other being base modification labile to hot piperidine. The investigation of the damaged sites and the inhibitory effects of radical scavengers revealed that hydroxyl radical was the species which attacked DNA in the reaction of H2O2/Fe(II). On the other hand, two types of DNA damage were induced by H2O2/Cu(II). Type I damage was predominant and inhibited by potassium iodide, but type II was not. The sites of the base-modification induced by type I damage were similar to those by lipid peroxidation products and by ascorbate in the presence of Cu(II), suggesting the involvement of radical species other than free hydroxyl radical in the damaging reactions.

Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites.

PubMed

Qin, Chunlin; Brunn, Jan C; Cook, Richard G; Orkiszewski, Ralph S; Malone, James P; Veis, Arthur; Butler, William T

2003-09-05

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.

Identification of interleukin-8 converting enzyme as cathepsin L.

PubMed

Ohashi, Kensaku; Naruto, Masanobu; Nakaki, Toshio; Sano, Emiko

2003-06-26

IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

Sequence-dependent DNA flexibility mediates DNase I cleavage.

PubMed

Heddi, Brahim; Abi-Ghanem, Josephine; Lavigne, Marc; Hartmann, Brigitte

2010-01-08

Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3' phosphate linkage flexibility, monitored by (31)P chemical shifts. Examining NMR-refined DNA structures highlighted that sequence-dependent flexible phosphates were associated with large minor groove variations that may promote the affinity of DNase I, according to relevant DNA-protein complexes. In sum, this work demonstrates that specificity in DNA-DNase I interaction is mediated by DNA flexibility, which influences the induced-fit transitions required to form productive complexes.

Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis

PubMed Central

Wang, Ning; Lee, I-Ju; Rask, Galen; Wu, Jian-Qiu

2016-01-01

The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis. PMID:27082518

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

PubMed Central

Mobley, E M; Pan, T

1999-01-01

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624

Mechanism of endonuclease cleavage by the HigB toxin

PubMed Central

Schureck, Marc A.; Repack, Adrienne; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

2016-01-01

Bacteria encode multiple type II toxin–antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro. Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition. PMID:27378776

Possible involvement of inefficient cleavage of preprovasopressin by signal peptidase as a cause for familial central diabetes insipidus.

PubMed Central

Ito, M; Oiso, Y; Murase, T; Kondo, K; Saito, H; Chinzei, T; Racchi, M; Lively, M O

1993-01-01

A transition of G to A at nucleotide position 279 in exon 1 of the vasopressin gene has been identified in patients with familial central diabetes insipidus. The mutation predicts an amino acid substitution of Thr (ACG) for Ala (GCG) at the COOH terminus of the signal peptide in preprovasopression (preproVP). Translation in vitro of wild-type and mutant mRNAs produced 19-kD preproVPs. When translated in the presence of canine pancreatic rough microsomes, wild-type preproVP was converted to a 21-kD protein, whereas the mutant mRNA produced proteins of 21 kD and 23 kD. NH2-terminal amino acid sequence analysis revealed that the 21-kD proteins from the wild-type and the mutants were proVPs generated by the proteolytic cleavage of the 19-residue signal peptide and the addition of carbohydrate. Accordingly, mutant preproVP was cleaved at the correct site after Thr-19, but the efficiency of cleavage by signal peptidase was < 25% that observed for the wild-type preproVP, resulting in the formation of a predominant glycosylated but uncleaved 23-kD product. These data suggest that inefficient processing of preproVP produced by the mutant allele is possibly involved in the pathogenesis of diabetes insipidus in the affected individuals. Images PMID:8514868

Identification of a Membrane Targeting and Degradation Signal in the p42 Protein of Influenza C Virus

PubMed Central

Pekosz, Andrew; Lamb, Robert A.

2000-01-01

Two mRNA species are derived from the influenza C virus RNA segment six, (i) a colinear transcript containing a 374-amino-acid residue open reading frame (referred to herein as the seg 6 ORF) which is translated to yield the p42 protein, and (ii) a spliced mRNA which encodes the influenza C virus matrix (CM1) protein consisting of the first 242 amino acids of p42. The p42 protein undergoes proteolytic cleavage at a consensus signal peptidase cleavage site after residue 259, yielding the p31 and CM2 proteins. Translocation of p42 into the endoplasmic reticulum membrane occurs cotranslationally and requires the hydrophobic internal signal peptide (residues 239 to 259), as well as the predicted transmembrane domain of CM2 (residues 285 to 308). The p31 protein was found to undergo rapid degradation after cleavage from p42. Addition of the 26S proteasome inhibitor lactacystin to influenza C virus-infected or seg 6 ORF cDNA-transfected cells drastically reduced p31 degradation. Transfer of the 17-residue C-terminal region of p31 to heterologous proteins resulted in their rapid turnover. The hydrophobic nature, but not the specific amino acid sequence of the 17-amino-acid C terminus of p31 appears to act as the signal for targeting the protein to membranes and for degradation. PMID:11044092

Primary genotypic resistance of HIV-1 to the maturation inhibitor PA-457 in protease inhibitor-experienced patients.

PubMed

Malet, Isabelle; Wirden, Marc; Derache, Anne; Simon, Anne; Katlama, Christine; Calvez, Vincent; Marcelin, Anne-Geneviève

2007-04-23

Sequences from 82 protease inhibitors (PI)-experienced patients were analysed for the presence of previously described in-vitro resistance mutations to PA-457 located in the C-terminal capside (H226Y, L231F, L231M) and in the N-terminal SP1 (A1V, A3T, A3V) within the CA-SP1 boundary domain. Overall, the CA-SP1 cleavage site was highly conserved in PI pre-treated patients and only one patient showed an L231M mutation. The impact of this mutation should be further addressed in vivo.

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

PubMed

Pane, Katia; Verrillo, Mariavittoria; Avitabile, Angela; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Di Maro, Antimo; Rega, Camilla; Amoresano, Angela; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

2018-04-18

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoB L , an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE

PubMed Central

Dunican, Brian F.; Hiller, David A.; Strobel, Scott A.

2015-01-01

The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active-site is atypical for acid-base catalysis, in that it is enriched for positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio-effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly pH dependent, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pKa of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pKa back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pKa shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites. PMID:26535789

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.

PubMed

Zheng, Nuoyan; Huang, Xiahe; Yin, Bojiao; Wang, Dan; Xie, Qi

2012-12-01

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

PubMed Central

Wood, Jeremy P.; Silveira, Jay R.; Maille, Nicole M.; Haynes, Laura M.

2011-01-01

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin. PMID:21131592

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

PubMed

Wood, Jeremy P; Silveira, Jay R; Maille, Nicole M; Haynes, Laura M; Tracy, Paula B

2011-02-03

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

PubMed Central

Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-01-01

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

PubMed Central

Beck, Zachary Q.; Lin, Ying-Chuan; Elder, John H.

2001-01-01

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors. PMID:11533208

Nuclear uptake of an amino-terminal fragment of apolipoprotein E4 promotes cell death and localizes within microglia of the Alzheimer's disease brain.

PubMed

Love, Julia E; Day, Ryan J; Gause, Justin W; Brown, Raquel J; Pu, Xinzhu; Theis, Dustin I; Caraway, Chad A; Poon, Wayne W; Rahman, Abir A; Morrison, Brad E; Rohn, Troy T

2017-01-01

Although harboring the apolipoprotein E4 ( APOE4 ) allele is a well known risk factor in Alzheimer's disease (AD), the mechanism by which it contributes to disease risk remains elusive. To investigate the role of proteolysis of apoE4 as a potential mechanism, we designed and characterized a site-directed cleavage antibody directed at position D151 of the mature form of apoE4 and E3. Characterization of this antibody indicated a high specificity for detecting synthesized recombinant proteins corresponding to the amino acid sequences 1-151 of apoE3 and E4 that would generate the 17 kDa (p17) fragment. In addition, this antibody also detected a ~17 kDa amino-terminal fragment of apoE4 following incubation with collagenase and matrix metalloproteinase-9 (MMP-9), but did not react with full-length apoE4. Application of this amino-terminal apoE cleavage-fragment (nApoECFp17) antibody, revealed nuclear labeling within glial cells and labeling of a subset of neurofibrillary tangles in the human AD brain. A quantitative analysis indicated that roughly 80% of labeled nuclei were microglia. To confirm these findings, cultured BV2 microglia cells were incubated with the amino-terminal fragment of apoE4 corresponding to the cleavage site at D151. The results indicated efficient uptake of this fragment and trafficking to the nucleus that also resulted in significant cell death. In contrast, a similarly designed apoE3 fragment showed no toxicity and primarily localized within the cytoplasm. These data suggest a novel cleavage event by which apoE4 is cleaved by the extracellular proteases, collagenase and MMP-9, generating an amino-terminal fragment that is then taken up by microglia, traffics to the nucleus and promotes cell death. Collectively, these findings provide important mechanistic insights into the mechanism by which harboring the APOE4 allele may elevate dementia risk observed in AD.

SfDredd, a Novel Initiator Caspase Possessing Activity on Effector Caspase Substrates in Spodoptera frugiperda

PubMed Central

Liu, Hao; Wu, Andong; Mei, Long; Liu, Qingzhen

2016-01-01

Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells. PMID:26977926

SfDredd, a Novel Initiator Caspase Possessing Activity on Effector Caspase Substrates in Spodoptera frugiperda.

PubMed

Yang, Zhouning; Zhou, Ke; Liu, Hao; Wu, Andong; Mei, Long; Liu, Qingzhen

2016-01-01

Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells.

[Markerless DNA deletion based on Red recombination and in vivo I-Sec I endonuclease cleavage in Escherichia coli chromosome].

PubMed

Zhu, Meiqin; Yu, Jian; Zhou, Changlin; Fang, Hongqing

2016-01-01

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.

Gravity and the orientation of cell division

NASA Technical Reports Server (NTRS)

Helmstetter, C. E.

1997-01-01

A novel culture system for mammalian cells was used to investigate division orientations in populations of Chinese hamster ovary cells and the influence of gravity on the positioning of division axes. The cells were tethered to adhesive sites, smaller in diameter than a newborn cell, distributed over a nonadhesive substrate positioned vertically. The cells grew and divided while attached to the sites, and the angles and directions of elongation during anaphase, projected in the vertical plane, were found to be random with respect to gravity. However, consecutive divisions of individual cells were generally along the same axis or at 90 degrees to the previous division, with equal probability. Thus, successive divisions were restricted to orthogonal planes, but the choice of plane appeared to be random, unlike the ordered sequence of cleavage orientations seen during early embryo development.

Bioinformatic investigation of the role of ubiquitins in cucumber flower morphogenesis

NASA Astrophysics Data System (ADS)

Pawełkowicz, Magdalena; Osipowski, Paweł; Wojcieszek, Michał; Kowalczuk, Cezary; PlÄ der, Wojciech; Przybecki, Zbigniew

2016-09-01

Three cDNA clones were used to screen cucumber genome in order to find genes and proteins. Functional annotation reveals that they are correlated with ubiquitination pathways. Various bioinformatics tools were used to screen and check protein sequences features such as: the presence of specific domains, transmembrane regions, cleavage site and cellular placement. The computational analysis for promotor region shows many binding sites for transcription factors, which could regulate the expression of genes. In order to check gene expression levels in developing flower buds of monoecious (B10) and gynoecious (2gg) cucumber lines, the real - time PCR technique was applied. The expression was checked for the whole buds and only for the 3rd and 4th whorls of bud when generative organ are form which were obtained by Laser Capture Microdissection (LCM) technique.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taneja, Bhupesh; Patel, Asmita; Slesarev, Alexei

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting nomore » conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.« less

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.

PubMed

Swarts, Daan C; van der Oost, John; Jinek, Martin

2017-04-20

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies. Copyright © 2017 Elsevier Inc. All rights reserved.