Base-CP proteasome can serve as a platform for stepwise lid formation

PubMed Central

Yu, Zanlin; Livnat-Levanon, Nurit; Kleifeld, Oded; Mansour, Wissam; Nakasone, Mark A.; Castaneda, Carlos A.; Dixon, Emma K.; Fushman, David; Reis, Noa; Pick, Elah; Glickman, Michael H.

2015-01-01

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes. PMID:26182356

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

PubMed Central

Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

2014-01-01

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit

PubMed Central

Ho, Jennifer Hei-Ngam; Kallstrom, George; Johnson, Arlen W.

2000-01-01

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway. PMID:11086007

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

PubMed Central

Russell, Jason D.; Scalf, Mark; Book, Adam J.; Ladror, Daniel T.; Vierstra, Richard D.; Smith, Lloyd M.; Coon, Joshua J.

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.

PubMed

Russell, Jason D; Scalf, Mark; Book, Adam J; Ladror, Daniel T; Vierstra, Richard D; Smith, Lloyd M; Coon, Joshua J

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway

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

Kwak, Mi-Kyoung; Kensler, Thomas W.

The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this genemore » was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the xenobiotic response elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed a blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway.« less

A gene duplication/loss event in the ribulose-1,5-bisphosphate-carboxylase/oxygenase (rubisco) small subunit gene family among accessions of Arabidopsis thaliana.

PubMed

Schwarte, Sandra; Tiedemann, Ralph

2011-06-01

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated.

Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation.

PubMed

Cukras, Anthony R; Green, Rachel

2005-05-27

The ribosomal protein S13 is found in the head region of the small subunit, where it interacts with the central protuberance of the large ribosomal subunit and with the P site-bound tRNA through its extended C terminus. The bridging interactions between the large and small subunits are dynamic, and are thought to be critical in orchestrating the molecular motions of the translation cycle. S13 provides a direct link between the tRNA-binding site and the movements in the head of the small subunit seen during translocation, thereby providing a possible pathway of signal transduction. We have created and characterized an rpsM(S13)-deficient strain of Escherichia coli and have found significant defects in subunit association, initiation and translocation through in vitro assays of S13-deficient ribosomes. Targeted mutagenesis of specific bridge and tRNA contact elements in S13 provides evidence that these two interaction domains play critical roles in maintaining the fidelity of translation. This ribosomal protein thus appears to play a non-essential, yet important role by modulating subunit interactions in multiple steps of the translation cycle.

Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

DOE PAGES

Dao, E. Han; Sierra, Raymond G.; Laksmono, Hartawan; ...

2015-04-30

In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecondmore » X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.« less

Deletion of Marek’s disease virus large subunit of ribonucleotide reductase (RR) impairs virus growth in vitro and in vivo

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens, called Marek’s disease (MD). In the unique long region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits o...

Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA.

PubMed

Hoshi, Toshinori; Tian, Yutao; Xu, Rong; Heinemann, Stefan H; Hou, Shangwei

2013-03-19

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are well known for their functional versatility, which is bestowed in part by their rich modulatory repertoire. We recently showed that long-chain omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) found in oily fish lower blood pressure by activating vascular BK channels made of Slo1+β1 subunits. Here we examined the action of DHA on BK channels with different auxiliary subunit compositions. Neuronal Slo1+β4 channels were just as well activated by DHA as vascular Slo1+β1 channels. In contrast, the stimulatory effect of DHA was much smaller in Slo1+β2, Slo1+LRRC26 (γ1), and Slo1 channels without auxiliary subunits. Mutagenesis of β1, β2, and β4 showed that the large effect of DHA in Slo1+β1 and Slo1+β4 is conferred by the presence of two residues, one in the N terminus and the other in the first transmembrane segment of the β1 and β4 subunits. Transfer of this amino acid pair from β1 or β4 to β2 introduces a large response to DHA in Slo1+β2. The presence of a pair of oppositely charged residues at the aforementioned positions in β subunits is associated with a large response to DHA. The Slo1 auxiliary subunits are expressed in a highly tissue-dependent fashion. Thus, the subunit composition-dependent stimulation by DHA demonstrates that BK channels are effectors of omega-3 fatty acids with marked tissue specificity.

Regulation of leaf organ size by the Arabidopsis RPT2a 19S proteasome subunit.

PubMed

Sonoda, Yutaka; Sako, Kaori; Maki, Yuko; Yamazaki, Naoko; Yamamoto, Hiroko; Ikeda, Akira; Yamaguchi, Junji

2009-10-01

The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins, to control many cellular events. To further understand this pathway, we focused on the RPT2 subunit of the 26S proteasome regulatory particle. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit, with a difference of only three amino acids in the protein sequences. Both genes showed similar mRNA accumulation patterns. However, the rpt2a mutant showed a specific phenotype of enlarged leaves caused by increased cell size, in correlation with increased ploidy. Detailed analyses revealed that cell expansion is increased in the rpt2a mutant by extended endoreduplication early in leaf development. The transcription of genes encoding cell cycle-related components, for DNA replication licensing and the G2/M phase, was also promoted in the rpt2a mutant, suggesting that extended endoreduplication was caused by increased DNA replication, and disrupted regulation of the G2/M checkpoint, at the proliferation stage of leaf development.

Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis

DOE PAGES

Marshall, Richard S.; Li, Faqiang; Gemperline, David C.; ...

2015-05-21

Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. In this paper, we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuingmore » proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Finally and collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes.« less

Reengineering ribosome export.

PubMed

Lo, Kai-Yin; Johnson, Arlen W

2009-03-01

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages.

Reengineering Ribosome Export

PubMed Central

Lo, Kai-Yin

2009-01-01

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages. PMID:19144820

Functional characterization of rpn3 uncovers a distinct 19S proteasomal subunit requirement for ubiquitin-dependent proteolysis of cell cycle regulatory proteins in budding yeast.

PubMed

Bailly, E; Reed, S I

1999-10-01

By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G(1)/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G(1)-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G(1) arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.

[Chymotripsin-like activity and subunit composition of proteasomes in human cancers].

PubMed

Kondakova, I V; Spirina, L V; Koval, V D; Shashova, E E; Choinzonov, E L; Ivanova, E V; Kolomiets, L A; Chernyshova, A L; Slonimskaya, E M; Usynin, E A; Afanasyev, S G

2014-01-01

Activity of the proteasome, polyfunctional enzymatic complex, is known to undergo changes during cancer development. This phenomenon is, probably, caused by the changes in subunit composition of proteasomes. In present work, we studied chymotrypsin-like activity of proteasomes, subunit composition and their association in breast cancer, head and neck squamous cell carcinoma, endometrial cancer, renal cancer, bladder cancer, stomach cancer and colorectal cancer. The increase of proteasome activity was revealed in most cancer tissues compared with adjacent tissues except for the renal cell carcinoma. Changes in proteasome activity in cancer tissues compared with correspondent normal tissues were accompanied by modification of its subunit composition. High proteasome activity was observed in combination with an increased expression of immune subunits and/or proteasome activator PA28, associated with activity of 20S proteasome. In breast cancer, head and neck squamous cell carcinoma, bladder cancer, stomach cancer and colorectal cancer we additionally found higher expression of Rpt6 subunit of 26S proteasome. Correlations between chymotrypsin like proteasome activity and subunit expressions were found in human cancer tissues. In summary, we suggest that proteasome ac- tivation and changes in its subunit composition plays an important role in cancer pathogenesis.

Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 60 S ribosomal subunit proteins L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39.

PubMed

Tsurugi, K; Collatz, E; Wool, E G; Lin, A

1976-12-25

The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Seventeen proteins (L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39) were isolated from three of the groups (B60, D60, G60) by ion exchange chromatography on carboxymethylcellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.5 to 15 mg. Eight of the proteins (L9, L11, L13, L21, L22, L35', L37 and L39) had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.

Marek’s Disease Virus Encoded Ribonucleotide Reductase Large Subunit is not Essential for In Vitro Replication

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) infected cells express a viral ribonucleotide reductase (RR) that is distinguishable from that present in uninfected cells by monoclonal antibody T81. Open reading frames UL39 and UL40 of the MDV genome encode the large (RR1) and small (RR2) subunits of RR enzyme, respe...

FAT10 knock out mice livers fail to develop Mallory-Denk bodies in the DDC mouse model.

PubMed

French, S W; French, B A; Oliva, J; Li, J; Bardag-Gorce, F; Tillman, B; Canaan, A

2012-12-01

Mallory-Denk bodies (MDBs) are aggresomes composed of undigested ubiqutinated short lived proteins which have accumulated because of a decrease in the rate of their degradation by the 26s proteasome. The decrease in the activity of the proteasome is due to a shift in the activity of the 26s proteasome to the immunoproteasome triggered by an increase in expression of the catalytic subunits of the immunoproteasome which replaces the catalytic subunits of the 26s proteasome. This switch in the type of proteasome in liver cells is triggered by the binding of IFNγ to the IFNγ sequence response element (ISRE) located on the FAT10 promoter. To determine if either FAT10 or IFNγ are essential for the formation of MDBs we fed both IFNγ and FAT10 knock out (KO) mice DDC added to the control diet for 10weeks in order to induce MDBs. Mice fed the control diet and Wild type mice fed the DDC or control diet were compared. MDBs were located by immunofluorescent double stains using antibodies to ubiquitin to stain MDBs and FAT10 to localize the increased expression of FAT10 in MDB forming hepatocytes. We found that MDB formation occurred in the IFNγ KO mice but not in the FAT10 KO mice. Western blots showed an increase in the ubiquitin smears and decreases β 5 (chymotrypsin-like 26S proteasome subunit) in the Wild type mice fed DDC but not in the FAT10 KO mice fed DDC. To conclude, we have demonstrated that FAT10 is essential to the induction of MDB formation in the DDC fed mice. Copyright © 2012 Elsevier Inc. All rights reserved.

CO2 directly modulates connexin 26 by formation of carbamate bridges between subunits

PubMed Central

Meigh, Louise; Greenhalgh, Sophie A; Rodgers, Thomas L; Cann, Martin J; Roper, David I; Dale, Nicholas

2013-01-01

Homeostatic regulation of the partial pressure of CO2 (PCO2) is vital for life. Sensing of pH has been proposed as a sufficient proxy for determination of PCO2 and direct CO2-sensing largely discounted. Here we show that connexin 26 (Cx26) hemichannels, causally linked to respiratory chemosensitivity, are directly modulated by CO2. A ‘carbamylation motif’, present in CO2-sensitive connexins (Cx26, Cx30, Cx32) but absent from a CO2-insensitive connexin (Cx31), comprises Lys125 and four further amino acids that orient Lys125 towards Arg104 of the adjacent subunit of the connexin hexamer. Introducing the carbamylation motif into Cx31 created a mutant hemichannel (mCx31) that was opened by increases in PCO2. Mutation of the carbamylation motif in Cx26 and mCx31 destroyed CO2 sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing of CO2. DOI: http://dx.doi.org/10.7554/eLife.01213.001 PMID:24220509

Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base.

PubMed

Funakoshi, Minoru; Tomko, Robert J; Kobayashi, Hideki; Hochstrasser, Mark

2009-05-29

The central protease of eukaryotes, the 26S proteasome, has a 20S proteolytic core particle (CP) and an attached 19S regulatory particle (RP). The RP is further subdivided into lid and base subcomplexes. Little is known about RP assembly. Here, we show that four conserved assembly factors govern biogenesis of the yeast RP base. Nas2 forms a complex with the Rpt4 and Rpt5 ATPases and enhances 26S proteasome formation in vivo and in vitro. Other RP subcomplexes contain Hsm3, which is related to mammalian proteasome subunit S5b. Hsm3 also contributes to base assembly. Larger Hsm3-containing complexes include two additional proteins, Nas6 and Rpn14, which function as assembly chaperones as well. Specific deletion combinations affecting these four factors cause severe perturbations to RP assembly. Our results demonstrate that proteasomal RP biogenesis requires multiple, functionally overlapping chaperones and suggest a model in which subunits form specific subcomplexes that then assemble into the base.

Marek’s disease virus encoded ribonucleotide reductase large subunit is essential for in vivo replication and plays a critical role in viral pathogenesis.

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus encodes a ribonucleotide reductase (RR) that consists of two subunits namely RR1 and RR2, both of which associate to form an active holoenzyme and both subunits are necessary for enzyme activity. It is an essential enzyme for the conversion of ribonucleotides to deoxyribonucleo...

Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

PubMed

Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

2015-07-01

The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Early cysteine-dependent inactivation of 26S proteasomes does not involve particle disassembly.

PubMed

Hugo, Martín; Korovila, Ioanna; Köhler, Markus; García-García, Carlos; Cabrera-García, J Daniel; Marina, Anabel; Martínez-Ruiz, Antonio; Grune, Tilman

2018-06-01

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

PubMed

Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

2015-01-13

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

The RPN5 subunit of the 26s proteasome is essential for gametogenesis, sporophyte development, and complex assembly in Arabidopsis.

PubMed

Book, Adam J; Smalle, Jan; Lee, Kwang-Hee; Yang, Peizhen; Walker, Joseph M; Casper, Sarah; Holmes, James H; Russo, Laura A; Buzzinotti, Zachri W; Jenik, Pablo D; Vierstra, Richard D

2009-02-01

The 26S proteasome is an essential multicatalytic protease complex that degrades a wide range of intracellular proteins, especially those modified with ubiquitin. Arabidopsis thaliana and other plants use pairs of genes to encode most of the core subunits, with both of the isoforms often incorporated into the mature complex. Here, we show that the gene pair encoding the regulatory particle non-ATPase subunit (RPN5) has a unique role in proteasome function and Arabidopsis development. Homozygous rpn5a rpn5b mutants could not be generated due to a defect in male gametogenesis. While single rpn5b mutants appear wild-type, single rpn5a mutants display a host of morphogenic defects, including abnormal embryogenesis, partially deetiolated development in the dark, a severely dwarfed phenotype when grown in the light, and infertility. Proteasome complexes missing RPN5a are less stable in vitro, suggesting that some of the rpn5a defects are caused by altered complex integrity. The rpn5a phenotype could be rescued by expression of either RPN5a or RPN5b, indicating functional redundancy. However, abnormal phenotypes generated by overexpression implied that paralog-specific functions also exist. Collectively, the data point to a specific role for RPN5 in the plant 26S proteasome and suggest that its two paralogous genes in Arabidopsis have both redundant and unique roles in development.

Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

PubMed Central

Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

2013-01-01

Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

Proteasome activity and their subunit composition in endometrial cancer tissue: correlations with clinical morphological parameters.

PubMed

Spirina, L V; Kondakova, I V; Koval', V D; Kolomiets, L A; Chernyshova, A L; Choinzonov, E L; Sharova, N P

2012-08-01

The development of endometrial cancer is related to the status of the intracellular proteasome system. Total proteasome activity and pools 26S and 20S activities are higher in tumor tissue than in intact endometrium, and their composition is different. The expression of α1α2α3α5α6α7 is lower in endometrial cancer tissue in comparison with intact endometrium and the content of immune subunits LMP7, LMP2, and PA28β is increased. Total proteasome activity depends on the disease stage.

Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Kojima, S; Kita, K

1997-01-01

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction. The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit. From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex. Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit. The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).

Phylogenetic relationships of the Gomphales based on nuc-25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined sequences

Treesearch

Admir J. Giachini; Kentaro Hosaka; Eduardo Nouhra; Joseph Spatafora; James M. Trappe

2010-01-01

Phylogenetic relationships among Geastrales, Gomphales, Hysterangiales, and Phallales were estimated via combined sequences: nuclear large subunit ribosomal DNA (nuc-25S-rDNA), mitochondrial small subunit ribosomal DNA (mit-12S-rDNA), and mitochondrial atp6 DNA (mit-atp6-DNA). Eighty-one taxa comprising 19 genera and 58 species...

Thermal- and urea-induced unfolding processes of glutathione S-transferase by molecular dynamics simulation.

PubMed

Li, Jiahuang; Chen, Yuan; Yang, Jie; Hua, Zichun

2015-05-01

The Schistosoma juponicum 26 kDa glutathione S-transferase (sj26GST) consists of the N-terminal domain (N-domain), containing three alpha-helices (named H1-H3) and four anti-parallel beta-strands (S1-S4), and the C-terminal domain (C-domain), comprising five alpha-helices (named H4-H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N-domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N-domain could not fold independent, suggesting that correct folding of N-domain depended on its interactions with C-domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C-domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C-domain and N-domain. The paper can offer insights into the molecular mechanism of GSTs unfolding. © 2014 Wiley Periodicals, Inc.

A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic

PubMed Central

2017-01-01

The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB. PMID:28154188

A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic.

PubMed

Gunn, Laura H; Valegård, Karin; Andersson, Inger

2017-04-21

The catalytic inefficiencies of the CO 2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L 2 ) 5 , and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L 8 S 8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L 8 S 8 enzymes between LSus of adjacent L 2 dimers, where negatively charged residues coordinate around a Mg 2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L 2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C 3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria.

PubMed

Glotz, C; Müssig, J; Gewitz, H S; Makowski, I; Arad, T; Yonath, A; Wittmann, H G

1987-11-01

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.

Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

PubMed Central

Umaer, Khan; Ciganda, Martin

2014-01-01

Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention. PMID:24706018

Subunit interface dynamics in hexadecameric rubisco.

PubMed

van Lun, Michiel; van der Spoel, David; Andersson, Inger

2011-09-02

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L(2) dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance. Copyright © 2011 Elsevier Ltd. All rights reserved.

The XPB subunit of repair/transcription factor TFIIH directly interacts with SUG1, a subunit of the 26S proteasome and putative transcription factor.

PubMed

Weeda, G; Rossignol, M; Fraser, R A; Winkler, G S; Vermeulen, W; van 't Veer, L J; Ma, L; Hoeijmakers, J H; Egly, J M

1997-06-15

Mutations in the basal transcription initiation/DNA repair factor TFIIH are responsible for three human disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS) and trichothiodystrophy (TTD). The non-repair features of CS and TTD are thought to be due to a partial inactivation of the transcription function of the complex. To search for proteins whose interaction with TFIIH subunits is disturbed by mutations in patients we used the yeast two-hybrid system and report the isolation of a novel XPB interacting protein, SUG1. The interaction was validated in vivo and in vitro in the following manner. (i) SUG1 interacts with XPB but not with the other core TFIIH subunits in the two-hybrid assay. (ii) Physical interaction is observed in a baculovirus co-expression system. (iii) In fibroblasts under non-overexpression conditions a portion of SUG1 is bound to the TFIIH holocomplex as deduced from co-purification, immunopurification and nickel-chelate affinity chromatography using functional tagged TFIIH. Furthermore, overexpression of SUG1 in normal fibroblasts induced arrest of transcription and a chromatin collapse in vivo. Interestingly, the interaction was diminished with a mutant form of XPB, thus providing a potential link with the clinical features of XP-B patients. Since SUG1 is an integral component of the 26S proteasome and may be part of the mediator, our findings disclose a SUG1-dependent link between TFIIH and the cellular machinery involved in protein modelling/degradation.

Variation in functional ascospore parts in the ascomycetous yeast Dipodascopsis uninucleata.

PubMed

Bareetseng, A S; Kock, J L F; Pohl, C H; Pretorius, E E; Van Wyk, P W J

2004-04-01

A variation in functional ascospore morphology was detected using electron microscopy (EM) in two varieties of the yeast Dipodascopsis uninucleata, i.e., D. uninucleata var. uninucleata and D. uninucleata var. wickerhamii. It was found that the latter produces ascospores characterized by the absence of small surface hooks which have been implicated in the release and re-assembly of ascospores in D. uninucleata var. uninucleata. These varieties are closely related on the basis of their mode of sexual reproduction, ascospore morphology as observed under the light microscope, physiological characteristics as well as the extent of divergence in the variable D1/D2 domain of the large subunit 26S ribosomal DNA.

Genetic homogeneity of Fascioloides magna in Austria.

PubMed

Husch, Christian; Sattmann, Helmut; Hörweg, Christoph; Ursprung, Josef; Walochnik, Julia

2017-08-30

The large American liver fluke, Fascioloides magna, is an economically relevant parasite of both domestic and wild ungulates. F. magna was repeatedly introduced into Europe, for the first time already in the 19th century. In Austria, a stable population of F. magna has established in the Danube floodplain forests southeast of Vienna. The aim of this study was to determine the genetic diversity of F. magna in Austria. A total of 26 individuals from various regions within the known area of distribution were investigated for their cytochrome oxidase subunit 1 (cox1) and nicotinamide dehydrogenase subunit 1 (nad1) gene haplotypes. Interestingly, all 26 individuals revealed one and the same haplotype, namely concatenated haplotype Ha5. This indicates a homogenous population of F. magna in Austria and may argue for a single introduction. Alternatively, genetic homogeneity might also be explained by a bottleneck effect and/or genetic drift. Copyright © 2017 Elsevier B.V. All rights reserved.

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

PubMed Central

Pathare, Ganesh Ramnath; Nagy, István; Bohn, Stefan; Unverdorben, Pia; Hubert, Agnes; Körner, Roman; Nickell, Stephan; Lasker, Keren; Sali, Andrej; Tamura, Tomohiro; Nishioka, Taiki; Förster, Friedrich; Baumeister, Wolfgang; Bracher, Andreas

2012-01-01

Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP. PMID:22187461

Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues.

PubMed

Wyroba, E; Surmacz, L; Osinska, M; Wiejak, J

2007-01-01

Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain

PubMed Central

Sakata, Eri; Yamaguchi, Yoshiki; Kurimoto, Eiji; Kikuchi, Jun; Yokoyama, Shigeyuki; Yamada, Shingo; Kawahara, Hiroyuki; Yokosawa, Hideyoshi; Hattori, Nobutaka; Mizuno, Yoshikuni; Tanaka, Keiji; Kato, Koichi

2003-01-01

Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, we determined the three-dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone 15N-1H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP. PMID:12634850

Estimating hydraulic parameters of a heterogeneous aquitard using long-term multi-extensometer and groundwater level data

NASA Astrophysics Data System (ADS)

Zhuang, Chao; Zhou, Zhifang; Illman, Walter A.; Guo, Qiaona; Wang, Jinguo

2017-09-01

The classical aquitard-drainage model COMPAC has been modified to simulate the compaction process of a heterogeneous aquitard consisting of multiple sub-units (Multi-COMPAC). By coupling Multi-COMPAC with the parameter estimation code PEST++, the vertical hydraulic conductivity ( K v) and elastic ( S ske) and inelastic ( S skp) skeletal specific-storage values of each sub-unit can be estimated using observed long-term multi-extensometer and groundwater level data. The approach was first tested through a synthetic case with known parameters. Results of the synthetic case revealed that it was possible to accurately estimate the three parameters for each sub-unit. Next, the methodology was applied to a field site located in Changzhou city, China. Based on the detailed stratigraphic information and extensometer data, the aquitard of interest was subdivided into three sub-units. Parameters K v, S ske and S skp of each sub-unit were estimated simultaneously and then were compared with laboratory results and with bulk values and geologic data from previous studies, demonstrating the reliability of parameter estimates. Estimated S skp values ranged within the magnitude of 10-4 m-1, while K v ranged over 10-10-10-8 m/s, suggesting moderately high heterogeneity of the aquitard. However, the elastic deformation of the third sub-unit, consisting of soft plastic silty clay, is masked by delayed drainage, and the inverse procedure leads to large uncertainty in the S ske estimate for this sub-unit.

Rpn9 Is Required for Efficient Assembly of the Yeast 26S Proteasome

PubMed Central

Takeuchi, Junko; Fujimuro, Masahiro; Yokosawa, Hideyosi; Tanaka, Keiji; Toh-e, Akio

1999-01-01

We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome. Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth. At the restrictive temperature, the Δrpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature. We analyzed the proteasome fractions separated by glycerol density gradient centrifugation by native polyacrylamide gel electrophoresis and found that a smaller amount of the 26S proteasome was produced in the Δrpn9 cells and that the 26S proteasome was shifted to lighter fractions than expected. The incomplete proteasome complexes were found to accumulate in the Δrpn9 cells. Furthermore, Rpn10 was not detected in the fractions containing proteasomes of the Δrpn9 cells. These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in the assembly and/or stability of the 26S proteasome. PMID:10490597

The ribosomes of Drosophila. II. Studies on intraspecific variation.

PubMed

Berger, E M; Weber, L

1974-12-01

Electrophoretic comparisons of 40S and 55S ribosomal subunit proteins from 18 strains of Drosophila melanogaster revealed the virtual absence of allelic variation. More detailed two-dimensional studies on the large subunit proteins in 6 of the strains demonstrated additional complexity but still no interstrain variation. The significance of these results is discussed with respect to present estimates of genic heterozygosity in natural populations.

Arginine 26 and aspartic acid 69 of the regulatory subunit are key residues of subunits interaction of acetohydroxyacid synthase isozyme III from E. coli.

PubMed

Zhao, Yuefang; Wen, Xin; Niu, Congwei; Xi, Zhen

2012-11-05

Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2.

PubMed

Whitney, Spencer Michael; Kane, Heather Jean; Houtz, Robert L; Sharwood, Robert Edward

2009-04-01

Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)8 and (S40L)16 oligomers that are devoid of unlinked S subunits. While there was little or no change in CO2/O2 specificity or carboxylation rate of the Rubisco oligomers, their Kms for CO2 and O2 were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called ANtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in ANtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO2-assimilation rates in ANtS40L at varying pCO2 corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

PubMed

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

The XPB subunit of repair/transcription factor TFIIH directly interacts with SUG1, a subunit of the 26S proteasome and putative transcription factor.

PubMed Central

Weeda, G; Rossignol, M; Fraser, R A; Winkler, G S; Vermeulen, W; van 't Veer, L J; Ma, L; Hoeijmakers, J H; Egly, J M

1997-01-01

Mutations in the basal transcription initiation/DNA repair factor TFIIH are responsible for three human disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS) and trichothiodystrophy (TTD). The non-repair features of CS and TTD are thought to be due to a partial inactivation of the transcription function of the complex. To search for proteins whose interaction with TFIIH subunits is disturbed by mutations in patients we used the yeast two-hybrid system and report the isolation of a novel XPB interacting protein, SUG1. The interaction was validated in vivo and in vitro in the following manner. (i) SUG1 interacts with XPB but not with the other core TFIIH subunits in the two-hybrid assay. (ii) Physical interaction is observed in a baculovirus co-expression system. (iii) In fibroblasts under non-overexpression conditions a portion of SUG1 is bound to the TFIIH holocomplex as deduced from co-purification, immunopurification and nickel-chelate affinity chromatography using functional tagged TFIIH. Furthermore, overexpression of SUG1 in normal fibroblasts induced arrest of transcription and a chromatin collapse in vivo. Interestingly, the interaction was diminished with a mutant form of XPB, thus providing a potential link with the clinical features of XP-B patients. Since SUG1 is an integral component of the 26S proteasome and may be part of the mediator, our findings disclose a SUG1-dependent link between TFIIH and the cellular machinery involved in protein modelling/degradation. PMID:9173976

Arabidopsis cop8 and fus4 mutations define the same gene that encodes subunit 4 of the COP9 signalosome.

PubMed Central

Serino, G; Tsuge, T; Kwok, S; Matsui, M; Wei, N; Deng, X W

1999-01-01

The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fus) mutants of Arabidopsis exhibit features of light-grown seedlings when grown in the dark. Cloning and biochemical analysis of COP9 have revealed that it is a component of a multiprotein complex, the COP9 signalosome (previously known as the COP9 complex). Here, we compare the immunoaffinity and the biochemical purification of the COP9 signalosome from cauliflower and confirm its eight-subunit composition. Molecular cloning of subunit 4 of the complex revealed that it is a proteasome-COP9 complex-eIF3 domain protein encoded by a gene that maps to chromosome 5, near the chromosomal location of the cop8 and fus4 mutations. Genetic complementation tests showed that the cop8 and fus4 mutations define the same locus, now designated as COP8. Molecular analysis of the subunit 4-encoding gene in both cop8 and fus4 mutants identified specific molecular lesions, and overexpression of the subunit 4 cDNA in a cop8 mutant background resulted in complete rescue of the mutant phenotype. Thus, we conclude that COP8 encodes subunit 4 of the COP9 signalosome. Examination of possible molecular interactions by using the yeast two-hybrid assay indicated that COP8 is capable of strong self-association as well as interaction with COP9, FUS6/COP11, FUS5, and Arabidopsis JAB1 homolog 1, the latter four proteins being previously defined subunits of the Arabidopsis COP9 signalosome. A comparative sequence analysis indicated that COP8 is highly conserved among multicellular eukaryotes and is also similar to a subunit of the 19S regulatory particle of the 26S proteasome. PMID:10521526

Evidence for the Location of the Allosteric Activation Switch in the Multisubunit Phosphorylase Kinase Complex from Mass Spectrometric Identification of Chemically Crosslinked Peptides*

PubMed Central

Nadeau, Owen W.; Anderson, David W.; Yang, Qing; Artigues, Antonio; Paschall, Justin E.; Wyckoff, Gerald J.; McClintock, Jennifer L.; Carlson, Gerald M.

2007-01-01

Phosphorylase kinase (PhK), an (αβγδ)4 complex, regulates glycogenolysis. Its activity, catalyzed by the γ subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory α, β and δ subunits. Activation of the catalytic γ subunit in the PhK complex by phosphorylation is known to be predominantly mediated by the regulatory β subunit, which undergoes a conformational change that is structurally linked with the γ subunit and that is characterized by the ability to form β-β dimers using a short chemical crosslinker. To determine potential regions of interaction of the β and γ subunits, we have used chemical crosslinking and 2-hybrid screening. The β and γ subunits were chemically crosslinked to each other in phosphorylated PhK, and crosslinked peptides were identified in digests of the kinase by Fourier transform mass spectrometry in combination with a search engine developed ‘in house’ that generates a hypothetical list of crosslinked peptides. Such a conjugate between β and γ was identified, verified by MS/MS and shown to correspond to crosslinking between K303 in the C-terminal regulatory domain of γ (γCRD) and R18 in the N-terminal regulatory region of β (β1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of β inhibited the crosslinking between β and γ in the complex, and was itself crosslinked to K303 of γ. Through the use of 2-hybrid screening, the β1-31 region was also shown to control β subunit self-interactions, which were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the β and γ subunits. The sum of our results considered together with previous findings implicates the γCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme’s regulatory subunits and is proximal to the active site cleft. PMID:17123541

Structure of ratcheted ribosomes with tRNAs in hybrid states

PubMed Central

Julián, Patricia; Konevega, Andrey L.; Scheres, Sjors H. W.; Lázaro, Melisa; Gil, David; Wintermeyer, Wolfgang; Rodnina, Marina V.; Valle, Mikel

2008-01-01

During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G. PMID:18971332

Insight into the transition between the open and closed conformations of Thermus thermophilus carboxypeptidase

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

Okai, Masahiko; Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477; Yamamura, Akihiro

Carboxypeptidase cleaves the C-terminal amino acid residue from proteins and peptides. Here, we report the functional and structural characterizations of carboxypeptidase belonging to the M32 family from the thermophilic bacterium Thermus thermophilus HB8 (TthCP). TthCP exhibits a relatively broad specificity for both hydrophilic (neutral and basic) and hydrophobic (aliphatic and aromatic) residues at the C-terminus and shows optimal activity in the temperature range of 75–80 °C and in the pH range of 6.8–7.2. Enzyme activity was significantly enhanced by cobalt or cadmium and was moderately inhibited by Tris at 25 °C. We also determined the crystal structure of TthCP at 2.6 Å resolution.more » Two dimer types of TthCP are present in the crystal. One type consists of two subunits in different states, open and closed, with a C{sup α} RMSD value of 2.2 Å; the other type consists of two subunits in the same open state. This structure enables us to compare the open and closed states of an M32 carboxypeptidase. The TthCP subunit can be divided into two domains, L and S, which are separated by a substrate-binding groove. The L and S domains in the open state are almost identical to those in the closed state, with C{sup α} RMSD values of 0.84 and 0.53 Å, respectively, suggesting that the transition between the open and closed states proceeds with a large hinge-bending motion. The superimposition between the closed states of TthCP and BsuCP, another M32 family member, revealed that most putative substrate-binding residues in the grooves are oriented in the same direction. - Highlights: • The enzyme activity of TthCP was inhibited moderately by Tris molecule. • We solved the crystal structure of TthCP at 2.6 Å resolution. • The crystal structure of TthCP revealed both the open and closed conformations.« less

A puzzle assembly strategy for fabrication of large engineered cartilage tissue constructs.

PubMed

Nover, Adam B; Jones, Brian K; Yu, William T; Donovan, Daniel S; Podolnick, Jeremy D; Cook, James L; Ateshian, Gerard A; Hung, Clark T

2016-03-21

Engineering of large articular cartilage tissue constructs remains a challenge as tissue growth is limited by nutrient diffusion. Here, a novel strategy is investigated, generating large constructs through the assembly of individually cultured, interlocking, smaller puzzle-shaped subunits. These constructs can be engineered consistently with more desirable mechanical and biochemical properties than larger constructs (~4-fold greater Young׳s modulus). A failure testing technique was developed to evaluate the physiologic functionality of constructs, which were cultured as individual subunits for 28 days, then assembled and cultured for an additional 21-35 days. Assembled puzzle constructs withstood large deformations (40-50% compressive strain) prior to failure. Their ability to withstand physiologic loads may be enhanced by increases in subunit strength and assembled culture time. A nude mouse model was utilized to show biocompatibility and fusion of assembled puzzle pieces in vivo. Overall, the technique offers a novel, effective approach to scaling up engineered tissues and may be combined with other techniques and/or applied to the engineering of other tissues. Future studies will aim to optimize this system in an effort to engineer and integrate robust subunits to fill large defects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Proteasomes remain intact, but show early focal alteration in their composition in a mouse model of amyotrophic lateral sclerosis.

PubMed

Kabashi, Edor; Agar, Jeffrey N; Hong, Yu; Taylor, David M; Minotti, Sandra; Figlewicz, Denise A; Durham, Heather D

2008-06-01

In amyotrophic lateral sclerosis caused by mutations in Cu/Zn-superoxide dismutase (SOD1), altered solubility and aggregation of the mutant protein implicates failure of pathways for detecting and catabolizing misfolded proteins. Our previous studies demonstrated early reduction of proteasome-mediated proteolytic activity in lumbar spinal cord of SOD1(G93A) transgenic mice, tissue particularly vulnerable to disease. The purpose of this study was to identify any underlying abnormalities in proteasomal structure. In lumbar spinal cord of pre-symptomatic mice [postnatal day 45 (P45) and P75], normal levels of structural 20S alpha subunits were incorporated into 20S/26S proteasomes; however, proteasomal complexes separated by native gel electrophoresis showed decreased immunoreactivity with antibodies to beta3, a structural subunit of the 20S proteasome core, and beta5, the subunit with chymotrypsin-like activity. This occurred prior to increase in beta5i immunoproteasomal subunit. mRNA levels were maintained and no association of mutant SOD1 with proteasomes was identified, implicating post-transcriptional mechanisms. mRNAs also were maintained in laser captured motor neurons at a later stage of disease (P100) in which multiple 20S proteins are reduced relative to the surrounding neuropil. Increase in detergent-insoluble, ubiquitinated proteins at P75 provided further evidence of stress on mechanisms of protein quality control in multiple cell types prior to significant motor neuron death.

Crystal structure of heterodimeric hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 reveals that the small subunit is directly involved in the product chain length regulation.

PubMed

Sasaki, Daisuke; Fujihashi, Masahiro; Okuyama, Naomi; Kobayashi, Yukiko; Noike, Motoyoshi; Koyama, Tanetoshi; Miki, Kunio

2011-02-04

Hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 (Ml-HexPPs) is a heterooligomeric type trans-prenyltransferase catalyzing consecutive head-to-tail condensations of three molecules of isopentenyl diphosphates (C(5)) on a farnesyl diphosphate (FPP; C(15)) to form an (all-E) hexaprenyl diphosphate (HexPP; C(30)). Ml-HexPPs is known to function as a heterodimer of two different subunits, small and large subunits called HexA and HexB, respectively. Compared with homooligomeric trans-prenyltransferases, the molecular mechanism of heterooligomeric trans-prenyltransferases is not yet clearly understood, particularly with respect to the role of the small subunits lacking the catalytic motifs conserved in most known trans-prenyltransferases. We have determined the crystal structure of Ml-HexPPs both in the substrate-free form and in complex with 7,11-dimethyl-2,6,10-dodecatrien-1-yl diphosphate ammonium salt (3-DesMe-FPP), an analog of FPP. The structure of HexB is composed of mostly antiparallel α-helices joined by connecting loops. Two aspartate-rich motifs (designated the first and second aspartate-rich motifs) and the other characteristic motifs in HexB are located around the diphosphate part of 3-DesMe-FPP. Despite the very low amino acid sequence identity and the distinct polypeptide chain lengths between HexA and HexB, the structure of HexA is quite similar to that of HexB. The aliphatic tail of 3-DesMe-FPP is accommodated in a large hydrophobic cleft starting from HexB and penetrating to the inside of HexA. These structural features suggest that HexB catalyzes the condensation reactions and that HexA is directly involved in the product chain length control in cooperation with HexB.

Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

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

Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi, E-mail: nomurat@shinshu-u.ac.jp

Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly,more » cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.« less

Characterization and analysis of posttranslational modifications of the human large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman sequencing.

PubMed

Odintsova, Tatyana I; Müller, Eva-Christina; Ivanov, Anton V; Egorov, Tsezi A; Bienert, Ralf; Vladimirov, Serguei N; Kostka, Susanne; Otto, Albrecht; Wittmann-Liebold, Brigitte; Karpova, Galina G

2003-04-01

The 60S ribosomal proteins were isolated from ribosomes of human placenta and separated by reversed phase HPLC. The fractions obtained were subjected to trypsin and Glu-C digestion and analyzed by mass fingerprinting (MALDI-TOF), MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found, 22 of which showed masses in accordance with the SwissProt database (June 2002) masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30, L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11, L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are consistent with N-terminal loss of methionine, acetylation, internal methylation, or hydroxylation. A loss of methionine without acetylation was found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19, L31, and L40), the molecular masses could not be determined. Proteins P1 and protein L3-like were not identified by the methods applied.

The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

PubMed Central

Bachnoff, Niv; Cohen-Kutner, Moshe; Atlas, Daphne

2011-01-01

A PKA consensus phosphorylation site S1928 at the α 11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α 11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α 11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α 11.2 or α 11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α 11.2, the pore forming subunit of CaV1.2. PMID:22216029

Structurally designed attenuated subunit vaccines for S. aureus LukS-PV and LukF-PV confer protection in a mouse bacteremia model.

PubMed

Karauzum, Hatice; Adhikari, Rajan P; Sarwar, Jawad; Devi, V Sathya; Abaandou, Laura; Haudenschild, Christian; Mahmoudieh, Mahta; Boroun, Atefeh R; Vu, Hong; Nguyen, Tam; Warfield, Kelly L; Shulenin, Sergey; Aman, M Javad

2013-01-01

Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens.

Isolation and characterization of cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits in Nitrosomonas sp. strain ENI-11.

PubMed

Hirota, Ryuichi; Kato, Junichi; Morita, Hiromu; Kuroda, Akio; Ikeda, Tsukasa; Takiguchi, Noboru; Ohtake, Hisao

2002-03-01

The cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large and small subunits in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 were cloned and sequenced. The deduced gene products, CbbL and CbbS, had 93 and 87% identity with Thiobacillus intermedius CbbL and Nitrobacter winogradskyi CbbS, respectively. Expression of cbbL and cbbS in Escherichia coli led to the detection of RubisCO activity in the presence of 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). To our knowledge, this is the first paper to report the genes involved in the carbon fixation reaction in chemolithotrophic ammonia-oxidizing bacteria.

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

PubMed

Yamaguchi, K; von Knoblauch, K; Subramanian, A R

2000-09-15

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

PubMed Central

Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C.; Hartl, F. Ulrich; Hayer-Hartl, Manajit

2015-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. PMID:25371207

Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome

PubMed Central

He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

2016-01-01

The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β. PMID:26683360

Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome.

PubMed

He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

2016-01-26

The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β.

Regulation of sGC via hsp90, Cellular Heme, sGC Agonists, and NO: New Pathways and Clinical Perspectives

PubMed Central

Ghosh, Arnab

2017-01-01

Abstract Significance: Soluble guanylate cyclase (sGC) is an intracellular enzyme that plays a primary role in sensing nitric oxide (NO) and transducing its multiple signaling effects in mammals. Recent Advances: The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells, including sGC, where it helps to drive heme insertion into the sGC-β1 subunit. This allows sGC-β1 to associate with a partner sGC-α1 subunit and mature into an NO-responsive active form. Critical Issues: In this article, we review evidence to date regarding the mechanisms that modulate sGC activity by a pathway where binding of hsp90 or sGC agonist to heme-free sGC dictates the assembly and fate of an active sGC heterodimer, both by NO and heme-dependent or heme-independent pathways. Future Directions: We discuss some therapeutic implications of the NO-sGC-hsp90 nexus and its potential as a marker of inflammatory disease. Antioxid. Redox Signal. 26, 182–190. PMID:26983679

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

PubMed Central

Bowman, Lisa; Flanagan, Lindsey; Fyfe, Paul K.; Parkin, Alison; Hunter, William N.; Sargent, Frank

2014-01-01

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function. PMID:24428762

Poly-Ub-Substrate-Degradative Activity of 26S Proteasome Is Not Impaired in the Aging Rat Brain

PubMed Central

Giannini, Carolin; Kloß, Alexander; Gohlke, Sabrina; Mishto, Michele; Nicholson, Thomas P.; Sheppard, Paul W.; Kloetzel, Peter-Michael; Dahlmann, Burkhardt

2013-01-01

Proteostasis is critical for the maintenance of life. In neuronal cells an imbalance between protein synthesis and degradation is thought to be involved in the pathogenesis of neurodegenerative diseases during aging. Partly, this seems to be due to a decrease in the activity of the ubiquitin-proteasome system, wherein the 20S/26S proteasome complexes catalyse the proteolytic step. We have characterised 20S and 26S proteasomes from cerebrum, cerebellum and hippocampus of 3 weeks old (young) and 24 month old (aged) rats. Our data reveal that the absolute amount of the proteasome is not dfferent between both age groups. Within the majority of standard proteasomes in brain the minute amounts of immuno-subunits are slightly increased in aged rat brain. While this goes along with a decrease in the activities of 20S and 26S proteasomes to hydrolyse synthetic fluorogenic tripeptide substrates from young to aged rats, the capacity of 26S proteasomes for degradation of poly-Ub-model substrates and its activation by poly-Ub-substrates is not impaired or even slightly increased in brain of aged rats. We conclude that these alterations in proteasome properties are important for maintaining proteostasis in the brain during an uncomplicated aging process. PMID:23667697

Polymerase ε1 mutation in a human syndrome with facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”)

PubMed Central

Pachlopnik Schmid, Jana; Lemoine, Roxane; Nehme, Nadine; Cormier-Daire, Valéry; Revy, Patrick; Debeurme, Franck; Debré, Marianne; Nitschke, Patrick; Bole-Feysot, Christine; Legeai-Mallet, Laurence; Lim, Annick; de Villartay, Jean-Pierre; Picard, Capucine; Durandy, Anne; Fischer, Alain

2012-01-01

DNA polymerase ε (Polε) is a large, four-subunit polymerase that is conserved throughout the eukaryotes. Its primary function is to synthesize DNA at the leading strand during replication. It is also involved in a wide variety of fundamental cellular processes, including cell cycle progression and DNA repair/recombination. Here, we report that a homozygous single base pair substitution in POLE1 (polymerase ε 1), encoding the catalytic subunit of Polε, caused facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”) in a large, consanguineous family. The mutation resulted in alternative splicing in the conserved region of intron 34, which strongly decreased protein expression of Polε1 and also to a lesser extent the Polε2 subunit. We observed impairment in proliferation and G1- to S-phase progression in patients’ T lymphocytes. Polε1 depletion also impaired G1- to S-phase progression in B lymphocytes, chondrocytes, and osteoblasts. Our results evidence the developmental impact of a Polε catalytic subunit deficiency in humans and its causal relationship with a newly recognized, inherited disorder. PMID:23230001

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

Spreitzer, Robert Joseph

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO 2 fixation in photosynthesis. However, it is a slow enzyme, and O 2 competes with CO 2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO 2. If carboxylation could be increased or oxygenation decreased, an increase in net CO 2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants,more » and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO 2/O 2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a possible structural pathway between the small-subunit βA-βB loop and alpha-helix 8 of the large-subunit α/β-barrel active site. Hybrid enzymes were also created comprised of plant small subunits and Chlamydomonas large subunits, and these enzymes have increases in CO 2/O 2 specificity, further indicating that small subunits may be the key for ultimately engineering an improved Rubisco enzyme.« less

Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Tachi, H; Yamamoto, K; Kumagai, H

1995-12-01

gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively. The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A gamma-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of gamma-glutamyltranspeptidase than the association of the separated large and small subunits.

Central cavity of fructose-1,6-bisphosphatase and the evolution of AMP/fructose 2,6-bisphosphate synergism in eukaryotic organisms.

PubMed

Gao, Yang; Shen, Lu; Honzatko, Richard B

2014-03-21

The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser(45) → His substantially fills the central cavity of pFBPase, and the triple mutation Ser(45) → His, Thr(46) → Arg, and Leu(186) → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.

Role of small subunit in mediating assembly of red-type form I Rubisco.

PubMed

Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C; Hartl, F Ulrich; Hayer-Hartl, Manajit

2015-01-09

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

PubMed

Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

2017-04-01

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two β subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C 24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. © 2017 Federation of European Biochemical Societies.

Structure of Ribosomal Silencing Factor Bound to Mycobacterium tuberculosis Ribosome.

PubMed

Li, Xiaojun; Sun, Qingan; Jiang, Cai; Yang, Kailu; Hung, Li-Wei; Zhang, Junjie; Sacchettini, James C

2015-10-06

The ribosomal silencing factor RsfS slows cell growth by inhibiting protein synthesis during periods of diminished nutrient availability. The crystal structure of Mycobacterium tuberculosis (Mtb) RsfS, together with the cryo-electron microscopy (EM) structure of the large subunit 50S of Mtb ribosome, reveals how inhibition of protein synthesis by RsfS occurs. RsfS binds to the 50S at L14, which, when occupied, blocks the association of the small subunit 30S. Although Mtb RsfS is a dimer in solution, only a single subunit binds to 50S. The overlap between the dimer interface and the L14 binding interface confirms that the RsfS dimer must first dissociate to a monomer in order to bind to L14. RsfS interacts primarily through electrostatic and hydrogen bonding to L14. The EM structure shows extended rRNA density that it is not found in the Escherichia coli ribosome, the most striking of these being the extended RNA helix of H54a. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome.

PubMed

Kisly, Ivan; Gulay, Suna P; Mäeorg, Uno; Dinman, Jonathan D; Remme, Jaanus; Tamm, Tiina

2016-05-22

During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote specific. These are mainly localized to the peripheral regions and are believed to stabilize the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here, the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 that is composed of an N-terminal globular domain, a middle region, and a long C-terminal α-helix. The analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilizing interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors, and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg(2+) concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasizing the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana

PubMed Central

Montacié, Charlotte; Durut, Nathalie; Opsomer, Alison; Palm, Denise; Comella, Pascale; Picart, Claire; Carpentier, Marie-Christine; Pontvianne, Frederic; Carapito, Christine; Schleiff, Enrico; Sáez-Vásquez, Julio

2017-01-01

In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity. PMID:29104584

Two Routes to Genetic Suppression of RNA Trimethylguanosine Cap Deficiency via C-Terminal Truncation of U1 snRNP Subunit Snp1 or Overexpression of RNA Polymerase Subunit Rpo26.

PubMed

Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

2015-04-24

The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme Tgs1 via sequential methyl additions to the N2 atom of the m(7)G cap. Whereas TMG caps are inessential for Saccharomyces cerevisiae vegetative growth at 25° to 37°, tgs1∆ cells that lack TMG caps fail to thrive at 18°. The cold-sensitive defect correlates with ectopic stoichiometric association of nuclear cap-binding complex (CBC) with the residual m(7)G cap of the U1 snRNA and is suppressed fully by Cbc2 mutations that weaken cap binding. Here, we show that normal growth of tgs1∆ cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the Snp1 subunit of yeast U1 snRNP. These results underscore the U1 snRNP as a focal point for TMG cap function in vivo. Casting a broader net, we conducted a dosage suppressor screen for genes that allowed survival of tgs1∆ cells at 18°. We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1∆ cold sensitivity, respectively. A structure-guided mutagenesis of Rpo26, using rpo26∆ complementation and tgs1∆ suppression as activity readouts, defined Rpo26-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for rpo26∆ complementation. The E124A and R135A alleles retained tgs1∆ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component. Copyright © 2015 Qiu et al.

Chronic aspirin via dose-dependent and selective inhibition of cardiac proteasome possibly contributed a potential risk to the ischemic heart.

PubMed

Tan, Chunjiang; Chen, Wenlie; Wu, Yanbin; Lin, Jiumao; Lin, Ruhui; Tan, Xuerui; Chen, Songming

2013-08-01

Impaired cardiac proteasome has been reported in ischemic heart and heart failure. Recent data highlighted aspirin as an inhibitor of the ubiquitin-proteasome system, however, it's unclear whether it affects cardiac proteasome functions. Myocardial infarction (MI), sham or normal male SD rats were injected intraperitoneally with high (300 mg/kg), low (5 mg/kg) aspirin or saline (control) once a day for seven weeks. Parallel experiments were performed in the hypoxia/reoxygenated human ventricular myocytes. Dose-related increases in heart and ventricular weight, and impaired cardiac functions, were found more exacerbated in the aspirin-treated MI rat hearts than the saline-treated MI counterparts. The activity of 26S, 20S and 19S declined by about 30%, or the 20S proteasome subunits β5, β2 and β1 decreased by 40%, 20% and 30%, respectively, in the MI rats compared with the non-MI rats (P<0.05). Compared with the saline-treated MI rats, 26S and 20S in high or low dose aspirin-treated MI rats further decreased by 30% and 20%, β5 by 30% and 12%, and β1 by 40% and 30%, respectively, and the lost activity was correlated with the compromised cardiac functions or the decreased cell viability. The dose-related and selective inhibition of 26S and 20S proteasome, or the 20S proteasome subunits β5 and β1 by aspirin was comparable to their protein expressions in the MI rats and in the cultured cells. The impaired cardiac proteasome, enhanced by chronic aspirin treatment, attenuated the removal of oxidative and ubiquitinated proteins, and chronic aspirin treatment via selective and dose-dependent inhibition of cardiac proteasome possibly constituted a potential risk to ischemic heart. Copyright © 2013 Elsevier Inc. All rights reserved.

Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis

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

Lee, Boo-Ja; Kwon, Sun Jae; Kim, Sung-Kyu

Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P{sub 0} interaction, but not during compatible TMV-P{sub 1.2} interaction. When CaRPN7::GFP fusion protein was targeted in onionmore » cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.« less

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

PubMed

Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon terminals. The type 2 neurons comprised 20% of the total population of alpha1-subunit-immunoreactive neurons. The remaining large-sized alpha1-immunoreactive cells were designated type 3 cells; they were positive for parvalbumin and were distinguished by long branching dendrites extending dorsally for 600-800 microm into the striatum. These neurons comprised 5% of the total population of alpha1-subunit-immunoreactive neurons and were surrounded by enkephalin-immunoreactive terminals. Electron microscopy showed that the alpha1-subunit type 3 neurons had an indented nuclear membrane and were densely covered with small axon terminals which established alpha1-subunit-immunoreactive symmetrical synaptic contacts with the soma and dendrites. These results provide a detailed characterization of the distribution, morphology and chemical characteristics of the alpha1-subunit-immunoreactive neurons in the rat striatum and suggest that the type 1 and type 2 neurons comprise of separate populations of striatal interneurons while the type 3 neurons may represent the large striatonigral projection neurons described by Bolam et al. [Bolam J. P., Somogyi P., Totterdell S. and Smith A. D. (1981) Neuroscience 6, 2141-2157.].

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency

PubMed Central

Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

2015-01-01

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. PMID:26208645

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency.

PubMed

Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

2015-11-01

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

PubMed Central

Occhipinti, Laura; Chang, Yiming; Altvater, Martin; Menet, Anna M.; Kemmler, Stefan; Panse, Vikram G.

2013-01-01

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel. PMID:23907389

S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

PubMed Central

Shipston, Michael J.

2014-01-01

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

Reaction mechanism of sterol hydroxylation by steroid C25 dehydrogenase - Homology model, reactivity and isoenzymatic diversity.

PubMed

Rugor, Agnieszka; Wójcik-Augustyn, Anna; Niedzialkowska, Ewa; Mordalski, Stefan; Staroń, Jakub; Bojarski, Andrzej; Szaleniec, Maciej

2017-08-01

Steroid C25 dehydrogenase (S25DH) is a molybdenum-containing oxidoreductase isolated from the anaerobic Sterolibacterium denitrificans Chol-1S. S25DH is classified as 'EBDH-like' enzyme (EBDH, ethylbenzene dehydrogenase) and catalyzes the introduction of an OH group to the C25 atom of a sterol aliphatic side-chain. Due to its regioselectivity, S25DH is proposed as a catalyst in production of pharmaceuticals: calcifediol or 25-hydroxycholesterol. The aim of presented research was to obtain structural model of catalytic subunit α and investigate the reaction mechanism of the O 2 -independent tertiary carbon atom activation. Based on homology modeling and theoretical calculations, a S25DH α subunit model was for the first time characterized and compared to other S25DH-like isoforms. The molecular dynamics simulations of the enzyme-substrate complexes revealed two stable binding modes of a substrate, which are stabilized predominantly by van der Waals forces in the hydrophobic substrate channel. However, H-bond interactions involving polar residues with C3=O/C3-OH in the steroid ring appear to be responsible for positioning the substrate. These results may explain the experimental kinetic results which showed that 3-ketosterols are hydroxylated 5-10-fold faster than 3-hydroxysterols. The reaction mechanism was studied using QM:MM and QM-only cluster models. The postulated mechanism involves homolytic CH cleavage by the MoO ligand, giving rise to a radical intermediate with product obtained in an OH rebound process. The hypothesis was supported by kinetic isotopic effect (KIE) experiments involving 25,26,26,26-[ 2 H]-cholesterol (4.5) and the theoretically predicted intrinsic KIE (7.0-7.2). Finally, we have demonstrated that the recombinant S25DH-like isoform catalyzes the same reaction as S25DH. Copyright © 2017 Elsevier Inc. All rights reserved.

Large subunit of the ribonucleotide reductase gene is a virulent factor and plays a critical role in Marek's disease virus pathogenesis

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR) gene consisting of two subunits UL39 (RR1) and UL40 (RR2). Both RR1 and RR2 form an active holoenzyme and are necessary for enzyme activity. This gene was indentified by monoclonal antibody T81 in a gt11 MDV expression library and f...

Crystal structure of human proteasome assembly chaperone PAC4 involved in proteasome formation.

PubMed

Kurimoto, Eiji; Satoh, Tadashi; Ito, Yuri; Ishihara, Eri; Okamoto, Kenta; Yagi-Utsumi, Maho; Tanaka, Keiji; Kato, Koichi

2017-05-01

The 26S proteasome is a large protein complex, responsible for degradation of ubiquinated proteins in eukaryotic cells. Eukaryotic proteasome formation is a highly ordered process that is assisted by several assembly chaperones. The assembly of its catalytic 20S core particle depends on at least five proteasome-specific chaperones, i.e., proteasome-assembling chaperons 1-4 (PAC1-4) and proteasome maturation protein (POMP). The orthologues of yeast assembly chaperones have been structurally characterized, whereas most mammalian assembly chaperones are not. In the present study, we determined a crystal structure of human PAC4 at 1.90-Å resolution. Our crystallographic data identify a hydrophobic surface that is surrounded by charged residues. The hydrophobic surface is complementary to that of its binding partner, PAC3. The surface also exhibits charge complementarity with the proteasomal α4-5 subunits. This will provide insights into human proteasome-assembling chaperones as potential anticancer drug targets. © 2017 The Protein Society.

The N-terminus of RPA large subunit and its spatial position are important for the 5'->3' resection of DNA double-strand breaks.

PubMed

Tammaro, Margaret; Liao, Shuren; McCane, Jill; Yan, Hong

2015-10-15

The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5' strand to generate 3' ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5'->3' directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA. We have found that the N-terminus of the RPA large subunit (RPA1N) interacts with both WRN and DNA2 and is essential for stimulating WRN's 3'->5' helicase activity and DNA2's 5'->3' ss-DNA exonuclease activity. A mutant RPA complex that lacks RPA1N is unable to support resection in Xenopus egg extracts and human cells. Furthermore, relocating RPA1N to the middle subunit but not to the small subunit causes severe defects in stimulating DNA2 and WRN and in supporting resection. Together, these findings suggest that RPA1N and its spatial position are critical for restricting the directionality of the WRN-DNA2 resection pathway. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Drosophila TGF-beta/Activin-like ligands Dawdle and Myoglianin appear to modulate adult lifespan through regulation of 26S proteasome function in adult muscle

PubMed Central

Langerak, Shaughna; Kim, Myung-Jun; Lamberg, Hannah; Godinez, Michael; Main, Mackenzie; Winslow, Lindsey; O'Connor, Michael B.

2018-01-01

ABSTRACT The Drosophila Activin signaling pathway employs at least three separate ligands – Activin-β (Actβ), Dawdle (Daw), and Myoglianin (Myo) – to regulate several general aspects of fruit fly larval development, including cell proliferation, neuronal remodeling, and metabolism. Here we provide experimental evidence indicating that both Daw and Myo are anti-ageing factors in adult fruit flies. Knockdown of Myo or Daw in adult fruit flies reduced mean lifespan, while overexpression of either ligand in adult muscle tissues but not in adipose tissues enhanced mean lifespan. An examination of ubiquitinated protein aggregates in adult muscles revealed a strong inverse correlation between Myo- or Daw-initiated Activin signaling and the amount of ubiquitinated protein aggregates. We show that this correlation has important functional implications by demonstrating that the lifespan extension effect caused by overexpression of wild-type Daw or Myo in adult muscle tissues can be completely abrogated by knockdown of a 26S proteasome regulatory subunit Rpn1 in adult fly muscle, and that the prolonged lifespan caused by overexpression of Daw or Myo in adult muscle could be due to enhanced protein levels of the key subunits of 26S proteasome. Overall, our data suggest that Activin signaling initiated by Myo and Daw in adult Drosophila muscles influences lifespan, in part, by modulation of protein homeostasis through either direct or indirect regulation of the 26S proteasome levels. Since Myo is closely related to the vertebrate muscle mass regulator Myostatin (GDF8) and the Myostatin paralog GDF11, our observations may offer a new experimental model for probing the roles of GDF11/8 in ageing regulation in vertebrates. This article has an associated First Person interview with the first author of the paper. PMID:29615416

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

PubMed Central

Klockenbusch, Cordula; Walsh, Geraldine M.; Brown, Lyda M.; Hoffman, Michael D.; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-01-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. PMID:25146974

ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase hexamer formation at low micromolar concentrations.

PubMed

Kuriata, Agnieszka M; Chakraborty, Manas; Henderson, J Nathan; Hazra, Suratna; Serban, Andrew J; Pham, Tuong V T; Levitus, Marcia; Wachter, Rebekka M

2014-11-25

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

PubMed Central

Lebreton, Alice; Saveanu, Cosmin; Decourty, Laurence; Rain, Jean-Christophe; Jacquier, Alain; Fromont-Racine, Micheline

2006-01-01

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation. PMID:16651379

Single channel properties of human α3 AChRs: impact of β2, β4 and α5 subunits

PubMed Central

Nelson, Mark E; Lindstrom, Jon

1999-01-01

We performed single channel analysis on human α3 acetylcholine receptors (AChRs) in Xenopus oocytes and native AChRs from the human neuroblastoma cell line IMR-32. α3 AChRs exhibit channel properties that reflect subunit composition.α3β2 AChR open times were 0.71 ± 0.14 and 3.5 ± 0.4 ms with a predominant conductance of 26 pS. α3β4 AChRs had open times of 1.4 ± 0.2 and 6.5 ± 0.8 ms and a predominant conductance of 31 pS. Burst times were 0.82 ± 0.12 and 5.3 ± 0.7 ms for α3β2 and 1.7 ± 0.1 and 16 ± 1 ms for α3β4. Desensitization was faster for AChRs with the β2 subunit than for those with the β4 subunit.One open time for α3α5β2 AChRs (5.5 ± 0.3 ms) was different from those of α3β2 AChRs. For α3α5β4 AChRs, an additional conductance, open time and burst time (36 pS, 22 ± 3 ms and 43 ± 4 ms, respectively) were different from those for α3β4 AChRs.α3 AChRs were inhibited by hexamethonium or mecamylamine. The rate constants for block of α3β4 by hexamethonium and of α3β2 by mecamylamine were 1.2 × 107 and 4.6 × 107 M−1 s−1, respectively.AChRs from IMR-32 cells had a predominant conductance of 32 pS and open times of 1.5 ± 0.3 and 9.6 ± 1.2 ms. These properties were most similar to those of α3β4 AChRs expressed in oocytes. Antibodies revealed that 5 ± 2% of IMR-32 α3 AChRs contained α5 subunits and 6 ± 2% contained β2 subunits. IMR-32 α3 AChRs are primarily α3β4 AChRs. PMID:10200416

Inhibition of the Host Proteasome Facilitates Papaya Ringspot Virus Accumulation and Proteosomal Catalytic Activity Is Modulated by Viral Factor HcPro

PubMed Central

Sahana, Nandita; Kaur, Harpreet; Basavaraj; Tena, Fatima; Jain, Rakesh Kumar; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2012-01-01

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant–pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro – PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation. PMID:23300704

Inhibition of the host proteasome facilitates papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HcPro.

PubMed

Sahana, Nandita; Kaur, Harpreet; Basavaraj; Tena, Fatima; Jain, Rakesh Kumar; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2012-01-01

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant-pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro - PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.

[A new mechanism of ubiquitin-dependent proteolytic pathway--polyubiquitin chain recognition and proteasomal targeting].

PubMed

Kawahara, Hiroyuki; Yokosawa, Hideyoshi

2008-01-01

The RPN10 subunit of 26S proteasome and several UBA domain proteins can bind to the polyubiquitin chain and play a role as ubiquitin receptors of the 26S proteasome. Although it was thought that substrate recognition is an essential step in the proteasome-mediated protein degradation, deletion of rpn10 genes in yeast does not influence the viability of cells but instead causes only a mild phenotype, suggesting that the above ubiquitin receptors are redundantly involved in substrate delivery to the proteasome. However, their functional difference is still enigmatic. In this review, we summarize recent advances in polyubiquitin chain recognition/delivery system and provide potential applications to modulate this system as a probable target for drug development.

GABAA receptors: Various stoichiometrics of subunit arrangement in α1β3 and α1β3ε receptors.

PubMed

Has, Ahmad Tarmizi Che; Chebib, Mary

2018-05-15

GABAA receptors (GABAARs) are members of the Cys-loop ligand-gated ion channel (LGIC) superfamily, which includes nicotinic acetylcholine, glycine, and serotonin (5HT3) receptors [1,2,3,4]. LGICs typically mediate fast synaptic transmission via the movement of ions through channels gated by neurotransmitters, such as acetylcholine for nicotinic receptors and GABA for GABAARs [5]. The term Cys-loop receptors originates from the presence of a conserved disulphide bond (or bridge) which holds together two cysteine amino acids of the loop that forms from the structure of polypeptides in the extracellular domain of the receptor's subunit [6]. GABAARs are pentameric transmembrane protein complexes consisting of five subunits from a variety of polypeptide subunits [7,8]. All of these subunits are pseudo-symmetrically organized in the plane of the membrane, with a Cl--selective channel in the middle of the complex. To date, nineteen GABAAR subunits have been identified and categorized into eight classes, α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3, but their variety is further broadened by the existence of several splice forms for certain subunits (e.g., α6, β2 and γ2) [9,10,11,12]. The subunits within each class have an amino acid sequence homology of 70% or more, whereas those with a sequence homology of 30% or less are grouped into different classes [13,14]. A subunit consists of four transmembrane domains (TM1-TM4), each forming an α-helix; a large extracellular N-terminal domain that incorporates part of the orthosteric agonist/antagonist binding site; a large intracellular loop between the TM3 and TM4; a small intracellular loop between TM1 and TM2; a small extracellular loop between TM2 and TM3; and a C-terminal extracellular domain [15,16]. Each subunit is arranged in such a way as to create principal and complementary interfaces with the other subunits, and in a position such that the TM2 of each subunit forms the wall of the channel pore [17,18,19]. The major subunit combination found in the brain comprises α1, β2 and γ2 subunits with the stoichiometry 2α1:2β2:1γ2 [18,20]. For the GABAA α1β2γ2 receptors, the subunits form a specific arrangement in which α1 and β2 subunits alternate with each other and are connected by a γ2 subunit (Figure A) [16,20,21]. For minor subtypes, different α and β subunits have been detected to co-exist as proven by the existence of GABAARs containing α1α2, α1α3, α1α5, α2α3, α3α5, α4α1, α4α2 and α4α3 in the central nervous system [22,23]. Meanwhile, the same pattern has been detected with β and γ subunits, at least the co-occurrence of β in the same GABAAR as well as γ2 with γ3, indicating that these subunits have the capacity to co-exist with each other [24,25,26]. Since different subunits can actually occur in one receptor, GABAARs are considered to exist in a multi-subunit arrangement, leading to ambiguity in the determination of a receptor's stoichiometry. In this review, we first briefly discuss the different subunit arrangements of α1 and β3 subunits in the binary α1β3 receptors. Then we review the GABAA ε-containing receptors predominantly in terms of the ability of ε subunit to present in different position in the ternary α1β3ε receptors, which introduce distinct populations of receptor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Sinophysis and Pseudophalacroma are distantly related to typical Dinophysoid dinoflagellates (Dinophysales, Dinophyceae).

PubMed

Gómez, Fernando; Moreira, David; López-García, Purificación

2012-01-01

Dinophysoid dinoflagellates are usually considered a large monophyletic group. Large subunit and small subunit (SSU) rDNA phylogenies suggest a basal position for Amphisoleniaceae (Amphisolenia,Triposolenia) with respect to two sister groups, one containing most Phalacroma species plus Oxyphysis and the other Dinophysis,Ornithocercus, Dinophysoid dinoflagellates are usually considered a large monophyletic group. Large subunit and small subunit (SSU) rDNA phylogenies suggest a basal position for Amphisoleniaceae (Amphisolenia,Triposolenia) with respect to two sister groups, one containing most Phalacroma species plus Oxyphysis and the other Dinophysis,Ornithocercus, Histioneis,Citharistes and some Phalacroma species. We provide here new SSU rDNA sequences of Pseudophalacroma (pelagic) and Sinophysis (the only benthic dinophysoid genus). Molecular phylogenies support that they are very divergent with respect to the main clade of Dinophysales. Additional molecular markers of these two key genera are needed to elucidate the evolutionary relations among the dinophysoid dinoflagellates. Histioneis,Citharistes and some Phalacroma species. We provide here new SSU rDNA sequences of Pseudophalacroma (pelagic) and Sinophysis (the only benthic dinophysoid genus). Molecular phylogenies support that they are very divergent with respect to the main clade of Dinophysales. Additional molecular markers of these two key genera are needed to elucidate the evolutionary relations among the dinophysoid dinoflagellates. © 2011 The Author(s) Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

Candida asparagi sp. nov., Candida diospyri sp. nov. and Candida qinlingensis sp. nov., novel anamorphic, ascomycetous yeast species.

PubMed

Lu, Hui-Zhong; Jia, Jian-Hua; Wang, Qi-Ming; Bai, Feng-Yan

2004-07-01

Among ascomycetous yeasts that were isolated from several nature reserve areas in China, three anamorphic strains isolated from soil (QL 5-5T) and fruit (QL 21-2T and SN 15-1T) were revealed, by conventional characterization and molecular phylogenetic analysis based on internal transcribed spacer and large subunit (26S) rRNA gene D1/D2 region sequencing, to represent three novel species in the genus Candida. Candida qinlingensis sp. nov. (type strain, QL 5-5T=AS 2.2524T=CBS 9768T) was related closely to a teleomorphic species, Williopsis pratensis. The close relatives of Candida diospyri sp. nov. (type strain, QL 21-2T=AS 2.2525T=CBS 9769T) are Candida friedrichii and Candida membranifaciens. Candida asparagi sp. nov. (type strain, SN 15-1T=AS 2.2526T=CBS 9770T) forms a clade with Candida fructus.

Central Cavity of Fructose-1,6-bisphosphatase and the Evolution of AMP/Fructose 2,6-bisphosphate Synergism in Eukaryotic Organisms*

PubMed Central

Gao, Yang; Shen, Lu; Honzatko, Richard B.

2014-01-01

The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser45 → His substantially fills the central cavity of pFBPase, and the triple mutation Ser45 → His, Thr46 → Arg, and Leu186 → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P. PMID:24436333

Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.

PubMed

He, Xi; Han, Ning; Wang, Yan-Ping

2016-01-01

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A{sub 2}

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

Santos, K. F.; Murakami, M. T.; Cintra, A. C. O.

2007-04-01

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A{sub 2} and a catalytically inactive acidic phospholipase A{sub 2} analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A{sub 2} and a catalytically inactive acidic phospholipase A{sub 2} analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained.more » The crotoxin complex crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 Å, and diffracted to 1.75 Å resolution. The crystal of the phospholipase A{sub 2} domain belongs to the hexagonal space group P6{sub 1}22 (or its enantiomorph P6{sub 5}22), with unit-cell parameters a = b = 38.7, c = 286.7 Å, and diffracted to 2.6 Å resolution. The crotapotin crystal diffracted to 2.3 Å resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.« less

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes.

PubMed

Stielow, J B; Lévesque, C A; Seifert, K A; Meyer, W; Iriny, L; Smits, D; Renfurm, R; Verkley, G J M; Groenewald, M; Chaduli, D; Lomascolo, A; Welti, S; Lesage-Meessen, L; Favel, A; Al-Hatmi, A M S; Damm, U; Yilmaz, N; Houbraken, J; Lombard, L; Quaedvlieg, W; Binder, M; Vaas, L A I; Vu, D; Yurkov, A; Begerow, D; Roehl, O; Guerreiro, M; Fonseca, A; Samerpitak, K; van Diepeningen, A D; Dolatabadi, S; Moreno, L F; Casaregola, S; Mallet, S; Jacques, N; Roscini, L; Egidi, E; Bizet, C; Garcia-Hermoso, D; Martín, M P; Deng, S; Groenewald, J Z; Boekhout, T; de Beer, Z W; Barnes, I; Duong, T A; Wingfield, M J; de Hoog, G S; Crous, P W; Lewis, C T; Hambleton, S; Moussa, T A A; Al-Zahrani, H S; Almaghrabi, O A; Louis-Seize, G; Assabgui, R; McCormick, W; Omer, G; Dukik, K; Cardinali, G; Eberhardt, U; de Vries, M; Robert, V

2015-12-01

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

PubMed Central

Erzberger, Jan P.; Stengel, Florian; Pellarin, Riccardo; Zhang, Suyang; Schaefer, Tanja; Aylett, Christopher H.S.; Cimermančič, Peter; Boehringer, Daniel; Sali, Andrej; Aebersold, Ruedi; Ban, Nenad

2014-01-01

Summary Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA. PMID:25171412

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

PubMed Central

Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

1996-01-01

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983

PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

NASA Technical Reports Server (NTRS)

Chen, B. Y.; Janes, H. W.; Gianfagna, T.

1998-01-01

Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

Subunit Rotavirus Vaccine Administered Parenterally to Rabbits Induces Active Protective Immunity

PubMed Central

Ciarlet, Max; Crawford, Sue E.; Barone, Christopher; Bertolotti-Ciarlet, Andrea; Ramig, Robert F.; Estes, Mary K.; Conner, Margaret E.

1998-01-01

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund’s adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 103 50% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P < 0.001, r = 0.55; Pearson’s correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund’s adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund’s adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund’s adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine. PMID:9765471

Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations.

PubMed Central

Nagata, K; Mizuta, T; Tonokatu, Y; Fukuda, Y; Okamura, H; Hayashi, T; Shimoyama, T; Tamura, T

1992-01-01

Monoclonal antibodies (MAbs) against the native urease of Helicobacter pylori NCTC 11637 were found to clearly inhibit the urease activity. Interestingly, synergistic inhibition by two MAbs recognizing different subunits was also observed. Ten MAbs were produced and classified as two isotypes of the immunoglobulin G (IgG) subclass, IgG1, and IgG2a. Western blot (immunoblot) analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that five MAbs recognized the large subunit and the other five recognized the small subunit of the urease. Among the MAbs, L2 and S2, which recognized the large and the small subunits, respectively, were also able to inhibit the urease activity of clinical isolates from H. pylori-infected patients. The combination of L2 and S2 led to augmented synergistic inhibition. L2, but not S2, could also inhibit the urease activity from Helicobacter mustelae; enzyme-linked immunosorbent assay and Western blot analysis showed that L2 cross-reacted with this urease. These results suggested that the epitope recognized by L2 had a structure common to both Helicobacter species and may be involved in the active site of the urease. In contrast to the MAbs, a polyclonal antibody in sera from mice immunized with H. pylori urease did not have the ability to inhibit H. pylori urease activity. However, the polyclonal antibody retained the ability to abolish the inhibitory action of these MAbs. Moreover, other MAbs which could not inhibit H. pylori urease activity also abolished the inhibitory action. Images PMID:1383158

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

Norris, J.R.; Budil, D.E.; Gast, P.

The orientation of the principal axes of the primary electron donor triplet state measured in single crystals of photosynthetic reaction centers is compared to the x-ray structures of the bacteria Rhodobacter (Rb.) sphaeroides R-26 and Rhodopseudomonas (Rps.) viridis. The primary donor of Rps. viridis is significantly different from that of Rb. sphaeroides. The measured directions of the axes indicate that triplet excitation is almost completely localized on the L-subunit half of the dimer in Rps. viridis but is more symmetrically distributed on the dimeric donor in Rb. sphaeroides R-26. The large reduction of the zero field splitting parameters relative tomore » monomeric bacteriochlorophyll triplet in vitro suggests significant participation of asymmetrical charge transfer electronic configurations in the special pair triplet state of both organisms.« less

Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host.

PubMed

Iwata, Fumiko; Shinjyo, Noriko; Amino, Hisako; Sakamoto, Kimitoshi; Islam, M Khyrul; Tsuji, Naotoshi; Kita, Kiyoshi

2008-03-01

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.

The Glucoamylase Inhibitor Acarbose Is a Direct Activator of Phosphorylase Kinase

PubMed Central

Nadeau, Owen W.; Liu, Weiya; Boulatnikov, Igor G.; Sage, Jessica M.; Peters, Jennifer L.; Carlson, Gerald M.

2011-01-01

Phosphorylase kinase (PhK), an (αβγδ)4 complex, stimulates energy production from glycogen in the cascade activation of glycogenolysis. Its large homologous α and β subunits regulate the activity of the catalytic γ subunit and account for 81% of PhK’s mass. Both subunits are thought to be multi-domain structures, and recent predictions based on their sequences suggest the presence of potentially functional glucoamylase (GH15)-like domains near their amino-termini. We present the first experimental evidence for such a domain in PhK, by demonstrating that the glucoamylase inhibitor acarbose binds PhK, perturbs its structure, and stimulates its kinase activity. PMID:20604537

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

PubMed

Klockenbusch, Cordula; Walsh, Geraldine M; Brown, Lyda M; Hoffman, Michael D; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-12-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

[Structure and function of eukaryotic nuclear DNA-dependent RNA polymerase I].

PubMed

Shematorova, E K; Shpakovskiĭ, G V

2002-01-01

In the eukaryotic cell, normal protein biosynthesis is sustained by several million ribosomes, which contain rRNA as an essential component. The high-molecular-weight precursor of large and 5.8S rRNAs is synthesized by DNA-dependent RNA polymerase I (Pol I) in the nucleolus. Data on DNA regulatory elements, protein factors involved in rDNA transcription by Pol I, subunit composition of Pol I, and on the interactions and possible functions of individual subunits are summarized.

The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

PubMed

Meister, Cindy; Gulko, Miriam Kolog; Köhler, Anna M; Braus, Gerhard H

2016-02-01

The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

Steric interactions lead to collective tilting motion in the ribosome during mRNA-tRNA translocation

NASA Astrophysics Data System (ADS)

Nguyen, Kien; Whitford, Paul C.

2016-02-01

Translocation of mRNA and tRNA through the ribosome is associated with large-scale rearrangements of the head domain in the 30S ribosomal subunit. To elucidate the relationship between 30S head dynamics and mRNA-tRNA displacement, we apply molecular dynamics simulations using an all-atom structure-based model. Here we provide a statistical analysis of 250 spontaneous transitions between the A/P-P/E and P/P-E/E ensembles. Consistent with structural studies, the ribosome samples a chimeric ap/P-pe/E intermediate, where the 30S head is rotated ~18°. It then transiently populates a previously unreported intermediate ensemble, which is characterized by a ~10° tilt of the head. To identify the origins of head tilting, we analyse 781 additional simulations in which specific steric features are perturbed. These calculations show that head tilting may be attributed to specific steric interactions between tRNA and the 30S subunit (PE loop and protein S13). Taken together, this study demonstrates how molecular structure can give rise to large-scale collective rearrangements.

Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits.

PubMed Central

Singh, S V; Partridge, C A; Awasthi, Y C

1984-01-01

Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively. Images Fig. 1. Fig. 5. PMID:6433888

Characterization of the Genes Encoding the Cytosolic and Plastidial Forms of ADP-Glucose Pyrophosphorylase in Wheat Endosperm1

PubMed Central

Burton, Rachel A.; Johnson, Philip E.; Beckles, Diane M.; Fincher, Geoffrey B.; Jenner, Helen L.; Naldrett, Mike J.; Denyer, Kay

2002-01-01

In most species, the synthesis of ADP-glucose (Glc) by the enzyme ADP-Glc pyrophosphorylase (AGPase) occurs entirely within the plastids in all tissues so far examined. However, in the endosperm of many, if not all grasses, a second form of AGPase synthesizes ADP-Glc outside the plastid, presumably in the cytosol. In this paper, we show that in the endosperm of wheat (Triticum aestivum), the cytosolic form accounts for most of the AGPase activity. Using a combination of molecular and biochemical approaches to identify the cytosolic and plastidial protein components of wheat endosperm AGPase we show that the large and small subunits of the cytosolic enzyme are encoded by genes previously thought to encode plastidial subunits, and that a gene, Ta.AGP.S.1, which encodes the small subunit of the cytosolic form of AGPase, also gives rise to a second transcript by the use of an alternate first exon. This second transcript encodes an AGPase small subunit with a transit peptide. However, we could not find a plastidial small subunit protein corresponding to this transcript. The protein sequence of the purified plastidial small subunit does not match precisely to that encoded by Ta.AGP.S.1 or to the predicted sequences of any other known gene from wheat or barley (Hordeum vulgare). Instead, the protein sequence is most similar to those of the plastidial small subunits from chickpea (Cicer arietinum) and maize (Zea mays) and rice (Oryza sativa) seeds. These data suggest that the gene encoding the major plastidial small subunit of AGPase in wheat endosperm has yet to be identified. PMID:12428011

Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Amino, H; Tomitsuka, E; Kita, K

1999-08-04

We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors.

PubMed

Zhang, Pengfei; Zawadzki, Robert J; Goswami, Mayank; Nguyen, Phuong T; Yarov-Yarovoy, Vladimir; Burns, Marie E; Pugh, Edward N

2017-04-04

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors' subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gα t ), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gα t the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s -1 Analyzing swelling as osmotically driven water influx, we find the H 2 O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10 -5 cm⋅s -1 , comparable to that of other cells lacking aquaporin expression. Application of Van't Hoff's law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H 2 O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.

Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

PubMed Central

Kwun, Hyun Jin; Shuda, Masahiro; Camacho, Carlos J.; Gamper, Armin M.; Thant, Mamie; Chang, Yuan

2015-01-01

ABSTRACT Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP), including PP2A A and C subunits and PP4C, as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of nonhuman polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT coimmunopurified with subunits PP2A Aα and PP2A C, MCV sT coimmunopurified with PP2A Aα, PP2A Aβ, and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV sT protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56α into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55α, B56α and B56ε into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth, but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting. IMPORTANCE Merkel cell polyomavirus is a newly discovered human cancer virus that promotes cancer, in part, through expression of its small T (sT) oncoprotein. Animal polyomavirus sT oncoproteins have been found to cause experimental tumors by blocking the activities of a group of phosphatases called protein phosphatase 2A (PP2A). Our structural analysis reveals that MCV sT also displaces the B subunit of PP2A to inhibit PP2A activity. MCV sT, however, only displaces a restricted subset of PP2A B subunits, which is insufficient to cause tumor cell formation in vitro. MCV sT instead transforms tumor cells through another region called the large T stabilization domain. The PP2A targeting and transforming activities lie on opposite faces of the MCV sT molecule and can be genetically separated from each other. PMID:25631078

Characterization and Evolution of Anthranilate 1,2-Dioxygenase from Acinetobacter sp. Strain ADP1

PubMed Central

Eby, D. Matthew; Beharry, Zanna M.; Coulter, Eric D.; Kurtz, Donald M.; Neidle, Ellen L.

2001-01-01

The two-component anthranilate 1,2-dioxygenase of the bacterium Acinetobacter sp. strain ADP1 was expressed in Escherichia coli and purified to homogeneity. This enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of O2 and consumption of one NADH. The terminal oxygenase component formed an α3β3 hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O2 consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O2 consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1,2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23°C) or nonpermissive (39°C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the α and β subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component. PMID:11114907

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.

PubMed

Sun, Linhan; Kao, Teh-Hui

2018-06-01

Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2 -haplotype and S 3 -haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCF SLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3 ) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2 ) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S 3 -RNase was completely suppressed by an antisense S 3 -RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

Developmentally regulated, alternative splicing of the Rpn10 gene generates multiple forms of 26S proteasomes

PubMed Central

Kawahara, Hiroyuki; Kasahara, Masanori; Nishiyama, Atsuya; Ohsumi, Keita; Goto, Tetsuya; Kishimoto, Takeo; Saeki, Yasushi; Yokosawa, Hideyoshi; Shimbara, Naoki; Murata, Shigeo; Chiba, Tomoki; Suzuki, Koichi; Tanaka, Keiji

2000-01-01

The 26S proteasome is a multisubunit protein- destroying machinery that degrades ubiquitin-tagged proteins. To date only a single species of Rpn10, which possibly functions as a multiubiquitin chain-binding subunit, has been identified in various organisms. Here we report that mouse Rpn10 mRNAs occur in at least five distinct forms, named Rpn10a to Rpn10e, and that they are generated from a single gene by developmentally regulated, alternative splicing. Rpn10a is ubiquitously expressed, whereas Rpn10e is expressed only in embryos, with the highest levels of expression in the brain. Both forms of Rpn10 are components of the 26S proteasome, with an apparently similar affinity for multiubiquitylated [125I]lysozyme in vitro. However, they exert markedly divergent effects on the destruction of B-type cyclin in Xenopus egg extracts. Thus, the 26S proteasome occurs in at least two functionally distinct forms: one containing a ubiquitously expressed Rpn10a and the other a newly identified, embryo-specific Rpn10e. While the former is thought to perform proteolysis constitutively in a wide variety of cells, the latter may play a specialized role in early embryonic development. PMID:10921894

Generic concepts in Nectriaceae

PubMed Central

Lombard, L.; van der Merwe, N.A.; Groenewald, J.Z.; Crous, P.W.

2015-01-01

The ascomycete family Nectriaceae (Hypocreales) includes numerous important plant and human pathogens, as well as several species used extensively in industrial and commercial applications as biodegraders and biocontrol agents. Members of the family are unified by phenotypic characters such as uniloculate ascomata that are yellow, orange-red to purple, and with phialidic asexual morphs. The generic concepts in Nectriaceae are poorly defined, since DNA sequence data have not been available for many of these genera. To address this issue we performed a multi-gene phylogenetic analysis using partial sequences for the 28S large subunit (LSU) nrDNA, the internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), the large subunit of the ATP citrate lyase (acl1), the RNA polymerase II largest subunit (rpb1), RNA polymerase II second largest subunit (rpb2), α-actin (act), β-tubulin (tub2), calmodulin (cmdA), histone H3 (his3), and translation elongation factor 1-alpha (tef1) gene regions for available type and authentic strains representing known genera in Nectriaceae, including several genera for which no sequence data were previously available. Supported by morphological observations, the data resolved 47 genera in the Nectriaceae. We re-evaluated the status of several genera, which resulted in the introduction of six new genera to accommodate species that were initially classified based solely on morphological characters. Several generic names are proposed for synonymy based on the abolishment of dual nomenclature. Additionally, a new family is introduced for two genera that were previously accommodated in the Nectriaceae. PMID:26955195

[Lectins from Sambucus nigra L inflorescences: isolation and investigation of biological activity using procaryotic test-systems].

PubMed

Karpova, I S; Korets'ka, N V; Pal'chykovs'ka, L H; Nehruts'ka, V V

2007-01-01

Isolation of lectins from extracts of the Sambucus nigra inflorescences and of pollen material have been performed using isoelectric focusing without carrier ampholytes (autofocusing). Fractions active in agglutination tests with different carbohydrate specificity were subjected to SDS-PAGE. The major lectin found in whole inflores-cences was GalNAc specific and is proposed to be a heterotetramer with subunits of about 30 and 33 kDa. It was called SNAflu-I. At least two other lectins were present in the pollen material and supposed to consist of identical subunits. Major positively charged lectin was Glc/Man specific with subunit of 26 kDa and called SNApol-I. Other pollen component (SNApol-II) was Gal specific with subunit of about 20 kDa. In order to elucidate cell targets sensitive for the S. nigra lectin's activity the combined effects of the lectins and transcriptional of phenazine origin on B. subtilis cells growth have been studied. Only SNApol-I demonstrated the antagonistic activity against these inhibitors in vivo. This lectin but not the SNAflu-I can also inhibit transcription in vitro. It is supposed that lectins from the same source may act in different directions on cell metabolism. Particularly one of the common targets may be the DNA-dependent synthesis of RNA.

RRP1B Targets PP1 to Mammalian Cell Nucleoli and Is Associated with Pre-60S Ribosomal Subunits

PubMed Central

Chamousset, Delphine; De Wever, Veerle; Moorhead, Greg B.; Chen, Yan; Boisvert, Francois-Michel; Lamond, Angus I.

2010-01-01

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions. PMID:20926688

Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

DOE PAGES

Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; ...

2014-12-22

Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is notmore » necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Finally, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.« less

Functional role of hCngb3 in regulation of human cone cng channel: effect of rod monochromacy-associated mutations in hCNGB3 on channel function.

PubMed

Okada, Akira; Ueyama, Hisao; Toyoda, Futoshi; Oda, Sanae; Ding, Wei-Guang; Tanabe, Shoko; Yamade, Shinichi; Matsuura, Hiroshi; Ohkubo, Iwao; Kani, Kazutaka

2004-07-01

The human cone photoreceptor cyclic nucleotide-gated (CNG) channel comprises alpha- and beta-subunits, which are respectively encoded by hCNGA3 and hCNGB3. The purpose was to examine the functional role of hCNGB3 in modulation of human cone CNG channels and to characterize functional consequences of rod monochromacy-associated mutations in hCNGB3 (S435F and D633G). Macroscopic patch currents were recorded from human embryonic kidney (HEK) 293 cells expressing homomeric (hCNGA3 and hCNGB3) and heteromeric (hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3-D633G) channels using inside-out patch-clamp technique. Both hCNGA3 homomeric and hCNGA3/hCNGB3 heteromeric channels were activated by cGMP, with half-maximally activating concentration (K(1/2)) of 11.1 +/- 1.0 and 26.2 +/- 1.9 micro M, respectively. The hCNGA3 channels appeared to be more sensitive to inhibition by extracellular Ca(2+) compared with hCNGA3/hCNGB3 channels, when assessed by the degree of outward rectification. Coexpression of either of rod monochromacy-associated mutants of hCNGB3 with hCNGA3 significantly reduced K(1/2) value for cGMP but little affected the sensitivity to extracellular Ca(2+), compared with wild-type heteromeric channels. The selectivity of hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3-D633G channels for monovalent cations were largely similar. Immunoprecipitation experiments showed association of hCNGA3 subunit with both of wild-type and mutant hCNGB3 subunits. The hCNGB3 plays an important modulatory role in the function of human cone CNG channels with respect to cGMP and extracellular Ca(2+) sensitivities. The rod monochromacy-associated S435F and D633G mutations in hCNGB3 evokes a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at least partly to pathogenesis of the disease.

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

PubMed Central

De Wachter, R; Neefs, J M; Goris, A; Van de Peer, Y

1992-01-01

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing. PMID:1561081

Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit.

PubMed

Haldar, Suranjana; Bevers, Loes E; Tosha, Takehiko; Theil, Elizabeth C

2011-07-22

Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O](n) multimers within the protein cage, en route to the cavity, at sites distributed over ~50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³⁺O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A(650 nm)) and on mineral growth (Fe³⁺O-A(350 nm)), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²⁺ access/selectivity to the active sites (Glu¹³⁰), (ii) distribution of Fe²⁺ to each of the three active sites near each ion channel (Asp¹²⁷), (iii) product (diferric oxo) release into the Fe³⁺O nucleation channels (Ala²⁶), and (iv) [Fe³⁺O](n) transit through subunits (Val⁴², Thr¹⁴⁹). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²⁺ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

PubMed Central

Liu, Guoxia; Zakharov, Sergey I.; Yao, Yongneng

2015-01-01

The large-conductance, voltage- and Ca2+-gated K+ (BK) channel consists of four α subunits, which form a voltage- and Ca2+-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca2+] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K+ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V50 for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50. PMID:25667410

Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

PubMed Central

Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

2007-01-01

Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

Dynamic contact network between ribosomal subunits enables rapid large-scale rotation during spontaneous translocation

PubMed Central

Bock, Lars V.; Blau, Christian; Vaiana, Andrea C.; Grubmüller, Helmut

2015-01-01

During ribosomal translation, the two ribosomal subunits remain associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. The absence of large barriers hindering rotation is a prerequisite for rapid rotation. Here, we investigate how such a flat free-energy landscape is achieved, in particular considering the large shifts the bridges undergo at the periphery. The dynamics and energetics of the intersubunit contact network are studied using molecular dynamics simulations of the prokaryotic ribosome in intermediate states of spontaneous translocation. Based on observed occupancies of intersubunit contacts, residues were grouped into clusters. In addition to the central contact clusters, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The tRNAs which span the subunits contribute to the intersubunit binding enthalpy to an almost constant degree, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation. PMID:26109353

Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

ERIC Educational Resources Information Center

Decatur, Wayne A.

2010-01-01

This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

PubMed

van Keulen, H; Campbell, S R; Erlandsen, S L; Jarroll, E L

1991-06-01

In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

Differential Phosphorylation of Plant Translation Initiation Factors by Arabidopsis thaliana CK2 Holoenzymes*

PubMed Central

Dennis, Michael D.; Browning, Karen S.

2009-01-01

A previously described wheat germ protein kinase (Yan, T. F., and Tao, M. (1982) J. Biol. Chem. 257, 7037–7043) was identified unambiguously as CK2 using mass spectrometry. CK2 is a ubiquitous eukaryotic protein kinase that phosphorylates a wide range of substrates. In previous studies, this wheat germ kinase was shown to phosphorylate eIF2α, eIF3c, and three large subunit (60 S) ribosomal proteins (Browning, K. S., Yan, T. F., Lauer, S. J., Aquino, L. A., Tao, M., and Ravel, J. M. (1985) Plant Physiol. 77, 370–373). To further characterize the role of CK2 in the regulation of translation initiation, Arabidopsis thaliana catalytic (α1 and α2) and regulatory (β1, β2, β3, and β4) subunits of CK2 were cloned and expressed in Escherichia coli. Recombinant A. thaliana CK2β subunits spontaneously dimerize and assemble into holoenzymes in the presence of either CK2α1 or CK2α2 and exhibit autophosphorylation. The purified CK2 subunits were used to characterize the properties of the individual subunits and their ability to phosphorylate various plant protein substrates. CK2 was shown to phosphorylate eIF2α, eIF2β, eIF3c, eIF4B, eIF5, and histone deacetylase 2B but did not phosphorylate eIF1, eIF1A, eIF4A, eIF4E, eIF4G, eIFiso4E, or eIFiso4G. Differential phosphorylation was exhibited by CK2 in the presence of various regulatory β-subunits. Analysis of A. thaliana mutants either lacking or overexpressing CK2 subunits showed that the amount of eIF2β protein present in extracts was affected, which suggests that CK2 phosphorylation may play a role in eIF2β stability. These results provide evidence for a potential mechanism through which the expression and/or subcellular distribution of CK2 β-subunits could participate in the regulation of the initiation of translation and other physiological processes in plants. PMID:19509278

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

PubMed Central

Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

2010-01-01

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation. PMID:20478262

Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication

DTIC Science & Technology

2007-03-01

proteasome. Mol. Cell, 6, 409–419. 14. Saeki, Y., Saitoh, A., Toh-e, A. & Yokosawa , H. (2002). Ubiquitin-like proteins and Rpn10 play cooperative...Sone, T., Toh-e, A. & Yokosawa , H. (2002). Identification of ubiquitin-like protein-binding sub- units of the 26 S proteasome. Biochem. Biophys. Res...Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nat Cell Biol 2002, 4:725-30. 15. Saeki Y, Sone T, Toh-e A, Yokosawa H: Identification of

A Novel Production Method for High-Fructose Glucose Syrup from Sucrose-Containing Biomass by a Newly Isolated Strain of Osmotolerant Meyerozyma guilliermondii.

PubMed

Khattab, Sadat Mohammad Rezq; Kodaki, Tsutomu

2016-04-28

One osmotolerant strain from among 44 yeast isolates was selected based on its growth abilities in media containing high concentrations of sucrose. This selected strain, named SKENNY, was identified as Meyerozyma guilliermondii by sequencing the internal transcribed spacer regions and partial D1/D2 large-subunit domains of the 26S ribosomal RNA. SK-ENNY was utilized to produce high-fructose glucose syrup (HFGS) from sucrose-containing biomass. Conversion rates to HFGS from 310-610 g/l of pure sucrose and from 75-310 g/l of sugar beet molasses were 73.5-94.1% and 76.2-91.1%, respectively. In the syrups produced, fructose yields were 89.4-100% and 96.5-100% and glucose yields were 57.6-82.5% and 55.3-79.5% of the theoretical values for pure sucrose and molasses sugars, respectively. This is the first report of employing M. guilliermondii for production of HFGS from sucrose-containing biomass.

Carney Complex: an update

PubMed Central

Correa, Ricardo; Salpea, Paraskevi; Stratakis, Constantine

2015-01-01

Carney Complex (CNC) is a rare autosomal dominant syndrome, characterized by pigmented lesions of the skin and mucosa, cardiac, cutaneous and other myxomas, and multiple endocrine tumors. The disease is caused by inactivating mutations or large deletions of the PRKAR1A gene located at 17q22–24 coding for the regulatory subunit type I alpha of protein kinase A (PKA) gene. Most recently, components of the complex have been associated with defects of other PKA subunits, such as the catalytic subunits PRKACA (adrenal hyperplasia) and PRKACB (pigmented spots, myxomas, pituitary adenomas). In this report, we review CNC, its clinical features, diagnosis, treatment, and molecular etiology including PRKAR1A mutations and the newest on PRKACA and PRKACB defects especially as they pertain to adrenal tumors and Cushing’s syndrome. PMID:26130139

The N-terminus of RPA large subunit and its spatial position are important for the 5′->3′ resection of DNA double-strand breaks

PubMed Central

Tammaro, Margaret; Liao, Shuren; McCane, Jill; Yan, Hong

2015-01-01

The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5′ strand to generate 3′ ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5′->3′ directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA. We have found that the N-terminus of the RPA large subunit (RPA1N) interacts with both WRN and DNA2 and is essential for stimulating WRN's 3′->5′ helicase activity and DNA2's 5′->3′ ss-DNA exonuclease activity. A mutant RPA complex that lacks RPA1N is unable to support resection in Xenopus egg extracts and human cells. Furthermore, relocating RPA1N to the middle subunit but not to the small subunit causes severe defects in stimulating DNA2 and WRN and in supporting resection. Together, these findings suggest that RPA1N and its spatial position are critical for restricting the directionality of the WRN-DNA2 resection pathway. PMID:26227969

Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions

PubMed Central

Graf, Michael; Arenz, Stefan; Huter, Paul; Dönhöfer, Alexandra; Nováček, Jiří

2017-01-01

Abstract Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA. PMID:27986857

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues.

PubMed

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia; Calzia, Daniela; Beitia, Maider; Morelli, Alessandro; Rosano, Camillo

2017-01-22

F 1 F o -ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H + flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H + within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F 1 and F O domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. Copyright © 2016 Elsevier Inc. All rights reserved.

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues

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

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia

F{sub 1}F{sub o}-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H{sup +} flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H{sup +} within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F{sub 1} andmore » F{sub O} domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.« less

Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2.

PubMed

Aigner, H; Wilson, R H; Bracher, A; Calisse, L; Bhat, J Y; Hartl, F U; Hayer-Hartl, M

2017-12-08

Plant RuBisCo, a complex of eight large and eight small subunits, catalyzes the fixation of CO 2 in photosynthesis. The low catalytic efficiency of RuBisCo provides strong motivation to reengineer the enzyme with the goal of increasing crop yields. However, genetic manipulation has been hampered by the failure to express plant RuBisCo in a bacterial host. We achieved the functional expression of Arabidopsis thaliana RuBisCo in Escherichia coli by coexpressing multiple chloroplast chaperones. These include the chaperonins Cpn60/Cpn20, RuBisCo accumulation factors 1 and 2, RbcX, and bundle-sheath defective-2 (BSD2). Our structural and functional analysis revealed the role of BSD2 in stabilizing an end-state assembly intermediate of eight RuBisCo large subunits until the small subunits become available. The ability to produce plant RuBisCo recombinantly will facilitate efforts to improve the enzyme through mutagenesis. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

PubMed

van Keulen, H; Gutell, R R; Campbell, S R; Erlandsen, S L; Jarroll, E L

1992-10-01

The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

PubMed Central

Ferrero, Rut; Torres, Magdalena

2002-01-01

Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage

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

Bae, Hee Kyong; Shinozuka, Junko; Islam, Zahidul

2009-06-01

Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultantmore » fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.« less

Purification and properties of the glutathione S-transferases from the anoxia-tolerant turtle, Trachemys scripta elegans.

PubMed

Willmore, William G; Storey, Kenneth B

2005-07-01

Glutathione S-transferases (GSTs) play critical roles in detoxification, response to oxidative stress, regeneration of S-thiolated proteins, and catalysis of reactions in nondetoxification metabolic pathways. Liver GSTs were purified from the anoxia-tolerant turtle, Trachemys scripta elegans. Purification separated a homodimeric (subunit relative molecular mass =34 kDa) and a heterodimeric (subunit relative molecular mass = 32.6 and 36.8 kDa) form of GST. The enzymes were purified 23-69-fold and 156-174-fold for homodimeric and heterodimeric GSTs, respectively. Kinetic data gathered using a variety of substrates and inhibitors suggested that both homodimeric and heterodimeric GSTs were of the alpha class although they showed significant differences in substrate affinities and responses to inhibitors. For example, homodimeric GST showed activity with known alpha class substrates, cumene hydroperoxide and p-nitrobenzylchloride, whereas heterodimeric GST showed no activity with cumene hydroperoxide. The specific activity of liver GSTs with chlorodinitrobenzene (CDNB) as the substrate was reduced by 2.6- and 8.7-fold for homodimeric and heterodimeric GSTs isolated from liver of anoxic turtles as compared with aerobic controls, suggesting an anoxia-responsive stable modification of the protein that may alter its function during natural anaerobiosis.

Identification and in vitro characterization of a Marek’s disease virus encoded ribonucleotide reductase

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR), a key regulatory enzyme in the DNA synthesis pathway. The gene coding for the RR of MDV is located in the unique long (UL) region of the genome. The large subunit is encoded by UL39 (RR1) and is predicted to comprise 860 amino acid...

Assembly and mechanism of a group II ECF transporter.

PubMed

Karpowich, Nathan K; Wang, Da-Neng

2013-02-12

Energy-coupling factor (ECF) transporters are a recently discovered family of primary active transporters for micronutrients and vitamins, such as biotin, thiamine, and riboflavin. Found exclusively in archaea and bacteria, including the human pathogens Listeria, Streptococcus, and Staphylococcus, ECF transporters may be the only means of vitamin acquisition in these organisms. The subunit composition of ECF transporters is similar to that of ATP binding cassette (ABC) importers, whereby both systems share two homologous ATPase subunits (A and A'), a high affinity substrate-binding subunit (S), and a transmembrane coupling subunit (T). However, the S subunit of ECF transporters is an integral membrane protein, and the transmembrane coupling subunits do not share an obvious sequence homology between the two transporter families. Moreover, the subunit stoichiometry of ECF transporters is controversial, and the detailed molecular interactions between subunits and the conformational changes during substrate translocation are unknown. We have characterized the ECF transporters from Thermotoga maritima and Streptococcus thermophilus. Our data suggests a subunit stoichiometry of 2S:2T:1A:1A' and that S subunits for different substrates can be incorporated into the same transporter complex simultaneously. In the first crystal structure of the A-A' heterodimer, each subunit contains a novel motif called the Q-helix that plays a key role in subunit coupling with the T subunits. Taken together, these findings suggest a mechanism for coupling ATP binding and hydrolysis to transmembrane transport by ECF transporters.

Role of Mex67-Mtr2 in the Nuclear Export of 40S Pre-Ribosomes

PubMed Central

Occhipinti, Laura; Kemmler, Stefan; Panse, Vikram G.

2012-01-01

Nuclear export of mRNAs and pre-ribosomal subunits (pre40S and pre60S) is fundamental to all eukaryotes. While genetic approaches in budding yeast have identified bona fide export factors for mRNAs and pre60S subunits, little is known regarding nuclear export of pre40S subunits. The yeast heterodimeric transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus and facilitates their passage through the nuclear pore complex (NPC) into the cytoplasm by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel. By exploiting a combination of genetic, cell-biological, and biochemical approaches, we uncovered an unanticipated role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes. We show that recruitment of Mex67-Mtr2 to pre40S subunits requires loops emanating from its NTF2-like domains and that the C-terminal FG-rich nucleoporin interacting UBA-like domain within Mex67 contributes to the transport of pre40S subunits to the cytoplasm. Remarkably, the same loops also recruit Mex67-Mtr2 to pre60S subunits and to the Nup84 complex, the respective interactions crucial for nuclear export of pre60S subunits and mRNAs. Thus Mex67-Mtr2 is a unique transport receptor that employs a common interaction surface to participate in the nuclear export of both pre-ribosomal subunits and mRNAs. Mex67-Mtr2 could engage a regulatory crosstalk among the three major export pathways for optimal cellular growth and proliferation. PMID:22956913

Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus

PubMed Central

Hull, J. Joe; Wang, Meixian

2014-01-01

The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits. PMID:26463065

Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes

PubMed Central

Xin Liu, Gong; Derst, Christian; Schlichthörl, Günter; Heinen, Steffen; Seebohm, Guiscard; Brüggemann, Andrea; Kummer, Wolfgang; Veh, Rüdiger W; Daut, Jürgen; Preisig-Müller, Regina

2001-01-01

The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea-pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea-pig cardiac ventricle. A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea-pig genomic library. The cell-specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi-cell RT-PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. After transfection in human embryonic kidney cells (HEK293) the mean single-channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. Cell-attached measurements in isolated guinea-pig cardiomyocytes (n = 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half-maximum block at -100 mV were 3.24 μm for gpKir2.1, 0.51 μm for gpKir2.2, 10.26 μm for gpKir2.3 and 235 μm for gpKir2.4. Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell-attached recordings. The concentration and voltage dependence of Ba2+ block of the large-conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. Our results suggest that the large-conductance inward rectifier channels found in guinea-pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate-conductance (23.8 pS) and low-conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively. PMID:11283229

In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae

PubMed Central

Li, Yong-Fu; Hess, Sonja; Pannell, Lewis K.; Tabor, Celia White; Tabor, Herbert

2001-01-01

S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form α and β subunits. The α subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based “suicide” inactivation. A large percentage of the α subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 ± 1 and m/z = 75 ± 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the α subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 ± 1 adduct, which is probably derived from the reaction product. Comparable modification of the α subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078–4081]. PMID:11526206

In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

PubMed

Li, Y F; Hess, S; Pannell, L K; White Tabor, C; Tabor, H

2001-09-11

S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].

Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

PubMed Central

Konikkat, Salini; Woolford, John L.

2017-01-01

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ~76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly. PMID:28062837

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.

Distinct cellular distributions of Kv4 pore-forming and auxiliary subunits in rat dorsal root ganglion neurons.

PubMed

Matsuyoshi, Hiroko; Takimoto, Koichi; Yunoki, Takakazu; Erickson, Vickie L; Tyagi, Pradeep; Hirao, Yoshihiko; Wanaka, Akio; Yoshimura, Naoki

2012-09-17

Dorsal root ganglia contain heterogeneous populations of primary afferent neurons that transmit various sensory stimuli. This functional diversity may be correlated with differential expression of voltage-gated K(+) (Kv) channels. Here, we examine cellular distributions of Kv4 pore-forming and ancillary subunits that are responsible for fast-inactivating A-type K(+) current. Expression pattern of Kv α-subunit, β-subunit and auxiliary subunit was investigated using immunohistochemistry, in situ hybridization and RT-PCR technique. The two pore-forming subunits Kv4.1 and Kv4.3 show distinct cellular distributions: Kv4.3 is predominantly in small-sized C-fiber neurons, whereas Kv4.1 is seen in DRG neurons in various sizes. Furthermore, the two classes of Kv4 channel auxiliary subunits are also distributed in different-sized cells. KChIP3 is the only significantly expressed Ca(2+)-binding cytosolic ancillary subunit in DRGs and present in medium to large-sized neurons. The membrane-spanning auxiliary subunit DPP6 is seen in a large number of DRG neurons in various sizes, whereas DPP10 is restricted in small-sized neurons. Distinct combinations of Kv4 pore-forming and auxiliary subunits may constitute A-type channels in DRG neurons with different physiological roles. Kv4.1 subunit, in combination with KChIP3 and/or DPP6, form A-type K(+) channels in medium to large-sized A-fiber DRG neurons. In contrast, Kv4.3 and DPP10 may contribute to A-type K(+) current in non-peptidergic, C-fiber somatic afferent neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2014-01-01

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

PubMed

Nedungadi, Divya; Binoy, Anupama; Pandurangan, Nanjan; Pal, Sanjay; Nair, Bipin G; Mishra, Nandita

2018-03-15

An α, β-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of β5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome†

PubMed Central

Wilson, Heather L.; Ou, Mark S.; Aldrich, Henry C.; Maupin-Furlow, Julie

2000-01-01

The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to ∼50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the α and β subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100°C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of β-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Δ1–73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80°C for the full-length protein to 65°C for PAN(Δ1–73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high Vmax for ATP and CTP hydrolysis of 3.5 and 5.8 μmol of Pi per min per mg of protein as well as a relatively low affinity for CTP and ATP with Km values of 307 and 497 μM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Δ1–73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that the M. jannaschii as well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction. PMID:10692374

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

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

Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying

2013-11-15

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletionmore » and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.« less

The β subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin

PubMed Central

Hanner, Markus; Schmalhofer, William A.; Munujos, Petraki; Knaus, Hans-Günther; Kaczorowski, Gregory J.; Garcia, Maria L.

1997-01-01

Transient expression of either α or α+β subunits of the high-conductance Ca2+-activated K+ (maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the α subunit. Although some properties of monoiodotyrosine-ChTX (125I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for 125I-ChTX measured under equilibrium binding conditions is increased ca. 50-fold in the presence of the β subunit. The rate constant for 125I-ChTX association is enhanced ca. 5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the β subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the β subunit has profound effects on 125I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop of β attract the positively charged 125I-ChTX to its binding site on α through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of β must contribute to stabilization of the toxin-bound state, either by a direct interaction with toxin, or through an allosteric effect on the α subunit. Certain regions in the extracellular loop of the β subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function. PMID:9096310

Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

PubMed Central

Stage-Zimmermann, Tracy; Schmidt, Ute; Silver, Pamela A.

2000-01-01

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ∼45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b–GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b–GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b–GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b–GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120. PMID:11071906

Soluble CD26 levels and its association to epidemiologic parameters in a sample population.

PubMed

De Chiara, Loretta; Rodríguez-Piñeiro, Ana M; Cordero, Oscar J; Rodríguez-Berrocal, Francisco J; Ayude, Daniel; Rivas-Hervada And, Francisco J; de la Cadena, María Páez

2009-01-01

Previous studies have suggested the use of soluble CD26 (sCD26) as a tumour marker for the detection of colorectal cancer (CRC) and advanced adenomas. The aim of this study was to assess the sCD26 concentration in a large cohort to evaluate its association to epidemiologic parameters and CRC-related symptoms/pathologies. Serum samples were collected from 2,754 putatively healthy individuals with ages ranging from 30-65 years, and with personal or familial history of polyps, CRC and/or CR symptoms. sCD26 levels were measured by ELISA. No association was found between the sCD26 concentration and age (< 50 and 50), the personal or familial history of polyps or CRC, rectal bleeding, haemorrhoids or diverticula. However, sCD26 was related to non-inflammatory benign pathologies (excluding rectal bleeding, changes in bowel habits, haemorrhoids, diverticula) and to inflammatory benign pathologies. Our results confirm that the sCD26 can be easily offered and evaluated in a large cohort. Additionally, the validation of sCD26 as a tumour marker for screening and case-finding purposes requires a further comparison with an established non-invasive test like the faecal occult blood.

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

PubMed Central

Shamayeva, Katsiaryna; Guzanova, Alena; Řeha, David; Csefalvay, Eva; Carey, Jannette; Weiserova, Marie

2017-01-01

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested. PMID:28133570

The extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity

PubMed Central

Pagliano, Cristina; La Rocca, Nicoletta; Andreucci, Flora; Deák, Zsuzsanna; Vass, Imre; Rascio, Nicoletta; Barbato, Roberto

2009-01-01

Background and Aims Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca2+ and Cl− as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl− and Ca2+, but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides. Methods Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT–PCR and time-resolved chlorophyll fluorescence were used. Key Results Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein. Conclusions In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII. PMID:19033288

Characterization of North American Armillaria species: Genetic relationships determined by ribosomal DNA sequences and AFLP markers

Treesearch

M. -S. Kim; N. B. Klopfenstein; J. W. Hanna; G. I. McDonald

2006-01-01

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS-1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data,...

Protection Conferred by recombinant Yersinia pestis Antigens Produced by a Rapid and Highly Scalable Plant Expression System

DTIC Science & Technology

2006-01-24

translational fusions with dsRED (lanes 8), and cytosol-targeted GFP (lanes 9). RbcL, large subunit of Rubisco . 862 ! www.pnas.org"cgi"doi൒.1073...analysis of F1-V expression with SDS"PAGE-Coomassie staining was difficult because the chimeric protein comigrates with the large subunit of Rubisco , a...contaminated by the Rubisco large subunit, which is very similar in size to F1-V. Analysis of Purified Plant-Produced Antigens. Western blots were

The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus.

PubMed

Weiss, Andy; Moore, Brittney D; Tremblay, Miguel H J; Chaput, Dale; Kremer, Astrid; Shaw, Lindsey N

2017-01-15

Staphylococcus aureus is a major human pathogen that causes infection in a wide variety of sites within the human body. Its ability to adapt to the human host and to produce a successful infection requires precise orchestration of gene expression. While DNA-dependent RNA polymerase (RNAP) is generally well characterized, the roles of several small accessory subunits within the complex have yet to be fully explored. This is particularly true for the omega (ω or RpoZ) subunit, which has been extensively studied in Gram-negative bacteria but largely neglected in Gram-positive counterparts. In Escherichia coli, it has been shown that ppGpp binding, and thus control of the stringent response, is facilitated by ω. Interestingly, key residues that facilitate ppGpp binding by ω are not conserved in S. aureus, and consequently, survival under starvation conditions is unaffected by rpoZ deletion. Further to this, ω-lacking strains of S. aureus display structural changes in the RNAP complex, which result from increased degradation and misfolding of the β' subunit, alterations in δ and σ factor abundance, and a general dissociation of RNAP in the absence of ω. Through RNA sequencing analysis we detected a variety of transcriptional changes in the rpoZ-deficient strain, presumably as a response to the negative effects of ω depletion on the transcription machinery. These transcriptional changes translated to an impaired ability of the rpoZ mutant to resist stress and to fully form a biofilm. Collectively, our data underline, for the first time, the importance of ω for RNAP stability, function, and cellular physiology in S. aureus IMPORTANCE: In order for bacteria to adjust to changing environments, such as within the host, the transcriptional process must be tightly controlled. Transcription is carried out by DNA-dependent RNA polymerase (RNAP). In addition to its major subunits (α 2 ββ') a fifth, smaller subunit, ω, is present in all forms of life. Although this small subunit is well studied in eukaryotes and Gram-negative bacteria, only limited information is available for Gram-positive and pathogenic species. In this study, we investigated the structural and functional importance of ω, revealing key roles in subunit folding/stability, complex assembly, and maintenance of transcriptional integrity. Collectively, our data underline, for the first time, the importance of ω for RNAP function and cellular harmony in S. aureus. Copyright © 2016 American Society for Microbiology.

Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system.

PubMed

Lee, J H; Maeda, S; Angelos, K L; Kamita, S G; Ramachandran, C; Walsh, D A

1992-11-03

Active gamma subunit of skeletal muscle phosphorylase kinase has been obtained by expression of the rat soleus cDNA in a baculovirus system. The protein exhibited the expected pH 6.8/8.2 activity ratio of 0.6, and its activity was insensitive to Ca2+ addition, indicating that it was free gamma subunit and not a gamma subunit-calmodulin complex. It was stimulated approximately 2-fold by Ca(2+)-calmodulin addition, demonstrating that it had retained high-affinity calmodulin binding. By site-directed mutagenesis, we have examined the role of six of the amino acids that constitute the consensus ATP binding site of the protein kinase, which in the gamma subunit is represented by the sequence 26Gly.Arg.Gly.Val.Ser.Ser.Val.Val33. Changes were evaluated by the kinetic determination of the dissociation constants of gamma-ATP, gamma-ADP, gamma-AMP.PCP, and gamma-phosphorylase and the maximum catalytic activity. The mutants Ser26-gamma, Ser29-gamma, Phe30-gamma, and Gly31-gamma each exhibited an essentially identical dissociation constant for gamma subunit phosphorylase, indicating that these mutations had not caused a global alteration in the protein structure but were limited to changes in the nucleotide binding site domain. Substitution of either Val33 (by Gly) or Gly28 (by Ser), two of the most conserved residues in all protein kinases, resulted in enzyme with marginally detectable activity. In noted contrast, the Ser26 mutant, which substituted the first glycine of the consensus glycine trio motif, and which is also very highly conserved, retained at least 25% of the enzymatic activity. The Gly31 substitution, which restored a glycine to a position characteristic for most protein kinases, had little overall effect upon the maximum rate of catalysis. Restoration of Ser30 to the more typical phenylalanine, which is present in most protein kinases, had minimal effect on catalysis. These data provide the first direct evaluation of the roles that different residues play within this consensus glycine trio/valine motif of the protein kinases, which up to now have only been surmised to be of importance because of their conservation. Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases (equivalent to Gly at position 31) does not produce a protein optimized for nucleotide binding.

Structural characterization of ribosome recruitment and translocation by type IV IRES.

PubMed

Murray, Jason; Savva, Christos G; Shin, Byung-Sik; Dever, Thomas E; Ramakrishnan, V; Fernández, Israel S

2016-05-09

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts tvhe otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.

Revisiting the structures of several antibiotics bound to the bacterial ribosome

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

Bulkley, David; Innis, C. Axel; Blaha, Gregor

2010-10-08

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model.more » Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.« less

Revisiting the Structures of Several Antibiotics Bound to the Bacterial Ribosome

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

D Bulkley; C Innis; G Blaha

2011-12-31

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model.more » Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.« less

Orientations and Proximities of the Extracellular Ends of Transmembrane Helices S0 and S4 in Open and Closed BK Potassium Channels

PubMed Central

Wu, Roland S.; Chudasama, Neelesh; Zakharov, Sergey I.; Karlin, Arthur; Marx, Steven O.

2013-01-01

The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the β1 subunit than in its absence, consistent with β1 acting through S0 to stabilize its immobilization relative to α S4. PMID:23472181

Candida amazonensis sp. nov., an ascomycetous yeast isolated from rotting wood in the Amazonian forest.

PubMed

Cadete, Raquel M; Melo, Monaliza A; Lopes, Mariana R; Pereira, Gilmara M D; Zilli, Jerri E; Vital, Marcos J S; Gomes, Fátima C O; Lachance, Marc-André; Rosa, Carlos A

2012-06-01

Five strains of a novel yeast species were isolated from rotting wood samples collected in an Amazonian forest site in the state of Roraima, northern Brazil. The sequences of the D1/D2 domains of the large subunit of the rRNA gene showed that this species belongs to the Scheffersomyces clade and is related to Candida coipomoensis, Candida lignicola and Candida queiroziae. The novel species Candida amazonensis sp. nov. is proposed to accommodate these isolates. The type strain of C. amazonensis sp. nov. is UFMG-HMD-26.3(T) ( = CBS 12363(T) = NRRL Y-48762(T)).

A simple cell-based high throughput screening (HTS) assay for inhibitors of Salmonella enterica RNA polymerase containing the general stress response regulator RpoS (σS).

PubMed

Campos-Gomez, Javier; Benitez, Jorge A

2018-07-01

RNA polymerase containing the stress response regulator σ S subunit (RpoS) plays a key role in bacterial survival in hostile environments in nature and during infection. Here we devise and validate a simple cell-based high throughput luminescence assay for this holoenzyme suitable for screening large chemical libraries in a robotic platform. Copyright © 2018 Elsevier B.V. All rights reserved.

Elastase-like Activity Is Dominant to Chymotrypsin-like Activity in 20S Proteasome's β5 Catalytic Subunit.

PubMed

Bensinger, Dennis; Neumann, Theresa; Scholz, Christoph; Voss, Constantin; Knorr, Sabine; Kuckelkorn, Ulrike; Hamacher, Kay; Kloetzel, Peter-Michael; Schmidt, Boris

2016-07-15

The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the β5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly β5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of β5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the β5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered.

γ-Aminobutyric Acid Type A α4, β2, and δ Subunits Assemble to Produce More Than One Functionally Distinct Receptor Type

PubMed Central

Eaton, Megan M.; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry

2014-01-01

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes. PMID:25238745

Studies on hydrogenase

PubMed Central

YAGI, Tatsuhiko; HIGUCHI, Yoshiki

2013-01-01

Hydrogenases are microbial enzymes which catalyze uptake and production of H2. Hydrogenases are classified into 10 classes based on the electron carrier specificity, or into 3 families, [NiFe]-family (including [NiFeSe]-subfamily), [FeFe]-family and [Fe]-family, based on the metal composition of the active site. H2 is heterolytically cleaved on the enzyme (E) to produce EHaHb, where Ha and Hb have different rate constants for exchange with the medium hydron. X-ray crystallography unveiled the three-dimensional structures of hydrogenases. The simplest [NiFe]-hydrogenase is a heterodimer, in which the large subunit bears the Ni-Fe center buried deep in the protein, and the small subunit bears iron-sulfur clusters, which mediate electron transfer between the Ni-Fe center and the protein surface. Some hydrogenases have additional subunit(s) for interaction with their electron carriers. Various redox states of the enzyme were characterized by EPR, FTIR, etc. Based on the kinetic, structural and spectroscopic studies, the catalytic mechanism of [NiFe]-hydrogenase was proposed to explain H2-uptake, H2-production and isotopic exchange reactions. PMID:23318679

The 19S proteasome activator promotes human cytomegalovirus immediate early gene expression through proteolytic and nonproteolytic mechanisms.

PubMed

Winkler, Laura L; Kalejta, Robert F

2014-10-01

Proteasomes are large, multisubunit complexes that support normal cellular activities by executing the bulk of protein turnover. During infection, many viruses have been shown to promote viral replication by using proteasomes to degrade cellular factors that restrict viral replication. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomal degradation of Daxx, a cellular transcriptional repressor that can silence viral immediate early (IE) gene expression. We previously showed that this degradation requires both the proteasome catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP associates with the 20S CP to facilitate protein degradation but also plays a 20S CP-independent role promoting transcription. Here, we present a nonproteolytic role of the 19S RP in HCMV IE gene expression. We demonstrate that 19S RP subunits are recruited to the major immediate early promoter (MIEP) that directs IE transcription. Depletion of 19S RP subunits generated a defect in RNA polymerase II elongation through the MIE locus during HCMV infection. Our results reveal that HCMV commandeers proteasome components for both proteolytic and nonproteolytic roles to promote HCMV lytic infection. Importance: Proteasome inhibitors decrease or eliminate 20S CP activity and are garnering increasing interest as chemotherapeutics. However, an increasing body of evidence implicates 19S RP subunits in important proteolytic-independent roles during transcription. Thus, pharmacological inhibition of the 20S CP as a means to modulate proteasome function toward therapeutic effect is an incomplete capitalization on the potential of this approach. Here, we provide an additional example of nonproteolytic 19S RP function in promoting HCMV transcription. These data provide a novel system with which to study the roles of different proteasome components during transcription, a rationale for previously described shifts in 19S RP subunit localization during HCMV infection, and a potential therapeutic intervention point at a pre-immediate early stage for the inhibition of HCMV infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Alpha B- and βA3-crystallins containing d-aspartic acids exist in a monomeric state.

PubMed

Sakaue, Hiroaki; Takata, Takumi; Fujii, Norihiko; Sasaki, Hiroshi; Fujii, Noriko

2015-01-01

Crystallin stability and subunit-subunit interaction are essential for eye lens transparency. There are three types of crystallins in lens, designated as α-, β-, and γ-crystallins. Alpha-crystallin is a hetero-polymer of about 800kDa, consisting of 35-40 subunits of two different αA- and αB-subunits, each of 20kDa. The β/γ-crystallin superfamily comprises oligomeric β-crystallin (2-6 subunits) and monomeric γ-crystallin. Since lens proteins have very long half-lives, they undergo numerous post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation, which may decrease crystallin solubility and ultimately cause cataract formation. Racemization and isomerization of aspartyl (Asp) residues have been detected only in polymeric α- and oligomeric β-crystallin, while the situation in monomeric γ-crystallin has not been studied. Here, we investigated the racemization and isomerization of Asp in the γ-crystallin fraction of elderly donors. The results show that Asp residues of γS-, γD- and γC-crystallins were not racemized and isomerized. However, strikingly, we found that a portion of αB-crystallin and βA3-crystallin moved to the lower molecular weight fraction which is the same size of γ-crystallin. In those fractions, Asp-96 of αB-crystallin and Asp-37 of βA3-crystallin were highly inverted, which do not occur in the native lens higher molecular weight fraction. Our results indicate the possibility that the inversion of Asp residues may induce dissociation of αB- and βA3-crystallins from the polymeric and oligomeric states. This is the first report that stereoinversion of amino acids disturbs lens protein assembly in aged human lens. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United Kingdom Mycology Reference Laboratory▿

PubMed Central

Linton, Christopher J.; Borman, Andrew M.; Cheung, Grace; Holmes, Ann D.; Szekely, Adrien; Palmer, Michael D.; Bridge, Paul D.; Campbell, Colin K.; Johnson, Elizabeth M.

2007-01-01

Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species. PMID:17251397

Modulation of Gain-of-function α6*-Nicotinic Acetylcholine Receptor by β3 Subunits*

PubMed Central

Dash, Bhagirathi; Lukas, Ronald J.

2012-01-01

We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9′ or 13′ positions (L9′S or V13′S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6(L9′S or V13′S)β4*- or α6(N143D+M145V)L9′Sβ2*-nAChR. This contrasts with the lack of consistent function when α6(L9′S or V13′S) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα6(N143D+M145V)L9′Shβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9′ or 13′ mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR. PMID:22315221

Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der.

PubMed

Hwang, Jihwan; Tseitin, Vladimir; Ramnarayan, Kal; Shenderovich, Mark D; Inouye, Masayori

2012-05-01

Der is an essential and widely conserved GTPase that assists assembly of a large ribosomal subunit in bacteria. Der associates specifically with the 50S subunit in a GTP-dependent manner and the cells depleted of Der accumulate the structurally unstable 50S subunit, which dissociates into an aberrant subunit at a lower Mg(2+) concentration. As Der is an essential and ubiquitous protein in bacteria, it may prove to be an ideal cellular target against which new antibiotics can be developed. In the present study, we describe our attempts to identify novel antibiotics specifically targeting Der GTPase. We performed the structure-based design of Der inhibitors using the X-ray crystal structure of Thermotoga maritima Der (TmDer). Virtual screening of commercially available chemical library retrieved 257 small molecules that potentially inhibit Der GTPase activity. These 257 chemicals were tested for their in vitro effects on TmDer GTPase and in vivo antibacterial activities. We identified three structurally diverse compounds, SBI-34462, -34566 and -34612, that are both biologically active against bacterial cells and putative enzymatic inhibitors of Der GTPase homologs. We also presented the possible interactions of each compound with the Der GTP-binding site to understand the mechanism of inhibition. Therefore, our lead compounds inhibiting Der GTPase provide scaffolds for the development of novel antibiotics against antibiotic-resistant pathogenic bacteria.

Phylogeographic patterns of Armillaria ostoyae in the western United States

Treesearch

J. W. Hanna; N. B. Klopfenstein; M. -S. Kim; G. I. McDonald; J. A. Moore

2007-01-01

Nuclear ribosomal DNA regions (i.e. large subunit, internal transcribed spacer, 5.8S and intergenic spacer) were sequenced using a direct-polymerase chain reaction method from Armillaria ostoyae genets collected from the western USA. Many of the A. ostoyae genets contained heterogeneity among rDNA repeats, indicating intragenomic variation and likely intraspecific...

Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

PubMed

Lewis, Brian A

2010-01-15

The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

Yeast Mitoribosome Large Subunit Assembly Proceeds by Hierarchical Incorporation of Protein Clusters and Modules on the Inner Membrane.

PubMed

Zeng, Rui; Smith, Erin; Barrientos, Antoni

2018-03-06

Mitoribosomes are specialized for the synthesis of hydrophobic membrane proteins encoded by mtDNA, all essential for oxidative phosphorylation. Despite their linkage to human mitochondrial diseases and the recent cryoelectron microscopy reconstruction of yeast and mammalian mitoribosomes, how they are assembled remains obscure. Here, we dissected the yeast mitoribosome large subunit (mtLSU) assembly process by systematic genomic deletion of 44 mtLSU proteins (MRPs). Analysis of the strain collection unveiled 37 proteins essential for functional mtLSU assembly, three of which are critical for mtLSU 21S rRNA stability. Hierarchical cluster analysis of mtLSU subassemblies accumulated in mutant strains revealed co-operative assembly of protein sets forming structural clusters and preassembled modules. It also indicated crucial roles for mitochondrion-specific membrane-binding MRPs in anchoring newly transcribed 21S rRNA to the inner membrane, where assembly proceeds. Our results define the yeast mtLSU assembly landscape in vivo and provide a foundation for studies of mitoribosome assembly across evolution. Copyright © 2018 Elsevier Inc. All rights reserved.

Allosteric Inhibition via R-state Destabilization in ATP Sulfurylase from Penicillium chrysogenum

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

MacRae, I. J.

2002-01-01

The structure of the cooperative hexameric enzyme ATP sulfurylase from Penicillium chrysogenum bound to its allosteric inhibitor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), was determined to 2.6 {angstrom} resolution. This structure represents the low substrate-affinity T-state conformation of the enzyme. Comparison with the high substrate-affinity R-state structure reveals that a large rotational rearrangement of domains occurs as a result of the R-to-T transition. The rearrangement is accompanied by the 17 {angstrom} movement of a 10-residue loop out of the active site region, resulting in an open, product release-like structure of the catalytic domain. Binding of PAPS is proposed to induce the allosteric transition bymore » destabilizing an R-state-specific salt linkage between Asp 111 in an N-terminal domain of one subunit and Arg 515 in the allosteric domain of a trans-triad subunit. Disrupting this salt linkage by site-directed mutagenesis induces cooperative inhibition behavior in the absence of an allosteric effector, confirming the role of these two residues.« less

Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

PubMed Central

Takahashi, Yuji; Shomura, Ayahiko; Sasaki, Takuji; Yano, Masahiro

2001-01-01

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of CK2α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype. PMID:11416158

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated.

PubMed

Jahangeer, S; Rodbell, M

1993-10-01

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process.

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated.

PubMed Central

Jahangeer, S; Rodbell, M

1993-01-01

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process. Images Fig. 1 Fig. 2 PMID:8415607

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in Escherichia coli.

PubMed

Koay, Teng Wei; Wong, Hann Ling; Lim, Boon Hoe

2016-11-26

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

Large G protein α-subunit XLαs limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo.

PubMed

He, Qing; Bouley, Richard; Liu, Zun; Wein, Marc N; Zhu, Yan; Spatz, Jordan M; Wang, Chia-Yu; Divieti Pajevic, Paola; Plagge, Antonius; Babitt, Jodie L; Bastepe, Murat

2017-11-07

Alterations in the activity/levels of the extralarge G protein α-subunit (XLαs) are implicated in various human disorders, such as perinatal growth retardation. Encoded by GNAS , XLαs is partly identical to the α-subunit of the stimulatory G protein (Gsα), but the cellular actions of XLαs remain poorly defined. Following an initial proteomic screen, we identified sorting nexin-9 (SNX9) and dynamins, key components of clathrin-mediated endocytosis, as binding partners of XLαs. Overexpression of XLαs in HEK293 cells inhibited internalization of transferrin, a process that depends on clathrin-mediated endocytosis, while its ablation by CRISPR/Cas9 in an osteocyte-like cell line (Ocy454) enhanced it. Similarly, primary cardiomyocytes derived from XLαs knockout (XLKO) pups showed enhanced transferrin internalization. Early postnatal XLKO mice showed a significantly higher degree of cardiac iron uptake than wild-type littermates following iron dextran injection. In XLKO neonates, iron and ferritin levels were elevated in heart and skeletal muscle, where XLαs is normally expressed abundantly. XLKO heart and skeletal muscle, as well as XLKO Ocy454 cells, showed elevated SNX9 protein levels, and siRNA-mediated knockdown of SNX9 in XLKO Ocy454 cells prevented enhanced transferrin internalization. In transfected cells, XLαs also inhibited internalization of the parathyroid hormone and type 2 vasopressin receptors. Internalization of transferrin and these G protein-coupled receptors was also inhibited in cells expressing an XLαs mutant missing the Gα portion, but not Gsα or an N-terminally truncated XLαs mutant unable to interact with SNX9 or dynamin. Thus, XLαs restricts clathrin-mediated endocytosis and plays a critical role in iron/transferrin uptake in vivo. Published under the PNAS license.

Chromatographic method for the isolation of active 40S and 60S subunits from rat liver polyribosomes.

PubMed

Kopacz-Jodczyk, T; Paszkiewicz-Gadek, A; Lopaczyński, W; Gałasiński, W

1984-04-06

A rapid and simple procedure for isolation of 40S and 60S ribosomal subunits by ion-exchange column chromatography is described. The dissociated ribosomes can be separated and non-ribosomal proteins and low-molecular-weight substances removed. An assessment by physicochemical and functional criteria showed that the ribosomal subunits obtained are active and sufficiently homogeneous.

The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

PubMed Central

Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

2012-01-01

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

Multi-perspective smFRET reveals rate-determining late intermediates of ribosomal translocation

PubMed Central

Wasserman, Michael R.; Alejo, Jose L.; Altman, Roger B.; Blanchard, Scott C.

2016-01-01

Directional translocation of the ribosome through the messenger RNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of transfer and messenger RNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we have tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations reveal direct evidence of structurally and kinetically distinct, late intermediates during substrate movement, whose resolution is rate-determining to the translocation mechanism. These steps involve intra-molecular events within the EFG(GDP)-bound ribosome, including exaggerated, reversible fluctuations of the small subunit head domain, which ultimately facilitate peptidyl-tRNA’s movement into its final post-translocation position. PMID:26926435

MUC1 and MUC4: Switching the Emphasis from Large to Small

PubMed Central

Carraway, Kermit L.

2011-01-01

Summation The MUC1 and MUC4 membrane mucins are each composed of a large alpha (α) and a small beta (β) subunit. The α subunits are fully exposed at the cell surface and contain variable numbers of repeated amino acid sequences that are heavily glycosylated. In contrast, the β subunits are much smaller and are anchored within the cell membrane, with their amino-terminal portions exposed at the cell surface and their carboxy-terminal tails facing the cytosol. Studies over the last several years are challenging the long-held belief that α subunits play the predominant role in cancer by conferring cellular properties that allow tumor cells to evade immune recognition and destruction. Indeed, the β subunits of MUC1 and MUC4 have emerged as oncogenes, as they engage signaling pathways responsible for tumor initiation and progression. Thus, a switch in the emphasis from the large α to the small β subunits offers attractive possibilities for successful clinical application. Such a focus shift is further supported by the absence of allelic polymorphism and variable glycosylation in the β subunit as well as by the presence of the β subunit in most MUC1 and MUC4 isoforms expressed by tumors. MUC1α, also known as CA15.3, is a Food and Drug Administration-approved serum biomarker for breast cancer, but its use is no longer recommended by the American Society of Clinical Oncology. However, comparison of β subunit expression in normal and malignant breast tissues may offer a novel approach to the exploitation of membrane mucins as biomarkers, as MUC1β-induced gene signatures with prognostic and predictive values in breast cancer have been reported. Preclinical studies with peptides that interfere with MUC1β oncogenic functions also look promising. PMID:21728842

Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors.

PubMed

Cameron, Krasnodara; Bartle, Emily; Roark, Ryan; Fanelli, David; Pham, Melissa; Pollard, Beth; Borkowski, Brian; Rhoads, Sarah; Kim, Joon; Rocha, Monica; Kahlson, Martha; Kangala, Melinda; Gentile, Lisa

2012-06-01

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design. Copyright © 2012 Elsevier Inc. All rights reserved.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. PMID:24934472

Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

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

Taylor, T.; Weintraub, B.D.

1985-04-01

The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing (/sup 14/C)alanine and (/sup 3/H) glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, (/supmore » 14/C)alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. (/sup 3/H)Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.« less

Structural characterization of ribosome recruitment and translocation by type IV IRES

PubMed Central

Murray, Jason; Savva, Christos G; Shin, Byung-Sik; Dever, Thomas E; Ramakrishnan, V; Fernández, Israel S

2016-01-01

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts the otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation. DOI: http://dx.doi.org/10.7554/eLife.13567.001 PMID:27159451

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

Structural and functional aspects of hetero-oligomers formed by the small heat shock proteins αB-crystallin and HSP27.

PubMed

Aquilina, J Andrew; Shrestha, Sudichhya; Morris, Amie M; Ecroyd, Heath

2013-05-10

αB-crystallin and HSP27 are mammalian intracellular small heat shock proteins. These proteins exchange subunits in a rapid and temperature-dependent manner. This facile subunit exchange suggests that differential expression could be used by the cell to regulate the response to stress. A robust technique defines parameters for the dynamic interaction between the major mammalian small heat shock proteins. Small heat shock proteins (sHSPs) exist as large polydisperse species in which there is constant dynamic subunit exchange between oligomeric and dissociated forms. Their primary role in vivo is to bind destabilized proteins and prevent their misfolding and aggregation. αB-Crystallin (αB) and HSP27 are the two most widely distributed and most studied sHSPs in the human body. They are coexpressed in different tissues, where they are known to associate with each other to form hetero-oligomeric complexes. In this study, we aimed to determine how these two sHSPs interact to form hetero-oligomers in vitro and whether, by doing so, there is an increase in their chaperone activity and stability compared with their homo-oligomeric forms. Our results demonstrate that HSP27 and αB formed polydisperse hetero-oligomers in vitro, which had an average molecular mass that was intermediate of each of the homo-oligomers and which were more thermostable than αB, but less so than HSP27. The hetero-oligomer chaperone function was found to be equivalent to that of αB, with each being significantly better in preventing the amorphous aggregation of α-lactalbumin and the amyloid fibril formation of α-synuclein in comparison with HSP27. Using mass spectrometry to monitor subunit exchange over time, we found that HSP27 and αB exchanged subunits 23% faster than the reported rate for HSP27 and αA and almost twice that for αA and αB. This represents the first quantitative evaluation of αB/HSP27 subunit exchange, and the results are discussed in the broader context of regulation of function and cellular proteostasis.

Crystal structures of bis-[(9S,13S,14S)-3-meth-oxy-17-methyl-morphinanium] tetra-chlorido-cobaltate and tetra-chlorido-cuprate.

PubMed

Gauchat, Eric; Nazarenko, Alexander Y

2017-01-01

(9 S ,13 S ,14 S )-3-Meth-oxy-17-methyl-morphinan (dextromethorphan) forms two isostructural salts with ( a ) tetra-chlorido-cobaltate, namely bis-[(9 S ,13 S ,14 S )-3-meth-oxy-17-methyl-morphinanium] tetra-chlorido-cobaltate, (C 18 H 26 NO) 2 [CoCl 4 ], and ( b ) tetra-chlorido-cuprate, namely bis-[(9 S ,13 S ,14 S )-3-meth-oxy-17-methyl-morphinanium] tetra-chlorido-cuprate, (C 18 H 26 NO) 2 [CuCl 4 ]. The distorted tetra-hedral anions are located on twofold rotational axes. The dextromethorphan cation can be described as being composed of two ring systems, a tetra-hydro-naphthalene system A + B and a deca-hydro-isoquinolinium subunit C + D , that are nearly perpendicular to one another: the angle between mean planes of the A + B and C + D moieties is 78.8 (1)° for ( a ) and 79.0 (1)° for ( b ). Two symmetry-related cations of protonated dextromethorphan are connected to the tetra-chlorido-cobaltate (or tetra-chlorido-cuprate) anions via strong N-H⋯Cl hydrogen bonds, forming neutral ion associates. These associates are packed in the (001) plane with no strong attractive bonding between them. Both compounds are attractive crystalline forms for unambiguous identification of the dextromethorphan and, presumably, of its optical isomer, levomethorphan.

A Monoclonal Antibody Toolkit for C. elegans

PubMed Central

Hadwiger, Gayla; Dour, Scott; Arur, Swathi; Fox, Paul; Nonet, Michael L.

2010-01-01

Background Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. Methodology/Principal Findings We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1), a component of synaptic vesicles; to Rim (UNC-10), a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1), a component of centrosomes; to CENP-C (HCP-4), which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2), a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5); to the nuclear envelope protein lamin (LMN-1); to EHD1 (RME-1) a marker for recycling endosomes; to caveolin (CAV-1), a marker for caveolae; to the cytochrome P450 (CYP-33E1), a resident of the endoplasmic reticulum; to β-1,3-glucuronyltransferase (SQV-8) that labels the Golgi; to a chaperonin (HSP-60) targeted to mitochondria; to LAMP (LMP-1), a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7) of the 26S proteasome; to dynamin (DYN-1) and to the α-subunit of the adaptor complex 2 (APA-2) as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1) and cadherin (HMR-1), both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1), which localized to apical membranes; to an ERBIN family protein (LET-413) which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7) which localizes to the plasma membrane at cell-cell contacts. In addition to working in whole mount immunocytochemistry, most of these antibodies work on western blots and thus should be of use for biochemical fractionation studies. Conclusions/Significance We have produced a set of monoclonal antibodies to subcellular components of the nematode C. elegans for the research community. These reagents are being made available through the Developmental Studies Hybridoma Bank (DSHB). PMID:20405020

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

PubMed

Body, A; Brownstein, B H

1978-01-01

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

PubMed Central

Body, Barbara A.; Brownstein, Bernard H.

1978-01-01

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction. PMID:412833

The role of TcdB and TccC subunits in secretion of the Photorhabdus Tcd toxin complex.

PubMed

Yang, Guowei; Waterfield, Nicholas R

2013-01-01

The Toxin Complex (TC) is a large multi-subunit toxin encoded by a range of bacterial pathogens. The best-characterized examples are from the insect pathogens Photorhabdus, Xenorhabdus and Yersinia. They consist of three large protein subunits, designated A, B and C that assemble in a 5∶1∶1 stoichiometry. Oral toxicity to a range of insects means that some have the potential to be developed as pest control technology. The three subunit proteins do not encode any recognisable export sequences and as such little progress has been made in understanding their secretion. We have developed heterologous TC production and secretion models in E. coli and used them to ascribe functions to different domains of the crucial B+C sub-complex. We have determined that the B and C subunits use a secretion mechanism that is either encoded by the proteins themselves or employ an as yet undefined system common to laboratory strains of E. coli. We demonstrate that both the N-terminal domains of the B and C subunits are required for secretion of the whole complex. We propose a model whereby the N-terminus of the C-subunit toxin exports the B+C sub-complex across the inner membrane while that of the B-subunit allows passage across the outer membrane. We also demonstrate that even in the absence of the B-subunit, that the C-subunit can also facilitate secretion of the larger A-subunit. The recognition of this novel export system is likely to be of importance to future protein secretion studies. Finally, the identification of homologues of B and C subunits in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggests that these toxins are likely to be important in a range of different hosts, including man.

Emulsifying properties of acidic subunits of soy 11S globulin.

PubMed

Liu, M; Lee, D S; Damodaran, S

1999-12-01

The emulsifying properties of the acidic subunits (AS11S) isolated from soy glycinin (11S) have been studied. The isolated AS11S existed in solution mainly as a dimer species. Circular dichroic analysis indicated only a slight increase in aperiodic structure and no significant difference in beta-sheet structure when compared with those of soy 11S. At similar experimental conditions, the emulsifying properties of AS11S were superior to those of soy 11S and heat-denatured 11S. Emulsions prepared with 1% AS11S remained very stable without any visible oil separation for more than a month under gentle agitating conditions, whereas those prepared with 1% 11S collapsed and separated into phases within 2-3 days. The AS11S-stabilized emulsions were very stable below 0.15 M ionic strength. Studies on the rate of adsorption and surface tension reduction at the air-water interface showed that AS11S was significantly more surface active than soy 11S. It is proposed that, because the mass fraction of acidic subunits in soy 11S is approximately 60% and it is relatively easy to separate the acidic subunits from soy 11S, it may be industrially feasible to develop an economical process to isolate functional acidic subunits for use in emulsion-based food products.

Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for diagnostic and therapeutic applications.

PubMed

Okolie, Charles Emeka; Cockayne, Alan; Penfold, Christopher; James, Richard

2013-11-19

Staphylococcus aureus produces several toxins, including Panton-Valentine leukocidin (PVL). The involvement of PVL in primary skin infections, necrotizing pneumonia, musculoskeletal disorders, brain abscess, and other diseases, some of which are life-threatening, has been reported. Following expert opinion, we aimed to provide the tools for establishment of sequence-based diagnostics and therapeutics for those conditions. We engineered the synergistic S and F (LukS-PV and LukF-PV respectively) pro-toxin subunits from Staphylococcus aureus USA400 into separate expression E. coli BL21(DE3)-pLysS hosts. Following Nickel affinity chromatography (NAC), the F subunit came out without bands of impurity. The S sub-unit did not come off very pure after NAC thus necessitating further purification by size exclusion and ion-exchange chromatography. The purification plots showed that the BioLogic-LP and AKTA systems are reliable for following the progress of the chromatographic purification in real-time. Computer predicted Mw for the 6His-LukF-PV and 6His-LukS-PV were 35645.41 Da and 33530.04 Da respectively, while the mass spectrometry results were 35643.57 Da and 33528.34 Da respectively. The BioLogic-LP and AKTA systems are commendable for reliability and user-friendliness. As a recent work elsewhere also reported that a second round of chromatography was necessary to purify the S subunit after the first attempt, we speculate that the S subunit might contain yet unidentified motif(s) requiring further treatment. The purified S and F sub-units of PVL were supplied to the Nottingham Cancer Immunotherapy group who used them to establish sequence-based monoclonal antibodies for diagnostic and therapeutic uses targeting PVL.

Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

PubMed

Kumar, Gokhlesh; Sarker, Subhodeep; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

2015-06-01

Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

PubMed Central

Walker, Sarah E.; Zhou, Fujun; Mitchell, Sarah F.; Larson, Victoria S.; Valasek, Leos; Hinnebusch, Alan G.; Lorsch, Jon R.

2013-01-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B’s domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome’s mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment. PMID:23236192

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

PubMed Central

Jakovljevic, Jelena; Ohmayer, Uli; Gamalinda, Michael; Talkish, Jason; Alexander, Lisa; Linnemann, Jan; Milkereit, Philipp; Woolford, John L.

2012-01-01

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA3 to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA3 pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A3 assembly factors and for proper assembly of the neighborhoods containing domains I and II. PMID:22893726

Characterization of a HoxEFUYH type of [NiFe] hydrogenase from Allochromatium vinosum and some EPR and IR properties of the hydrogenase module.

PubMed

Long, Minnan; Liu, Jingjing; Chen, Zhifeng; Bleijlevens, Boris; Roseboom, Winfried; Albracht, Simon P J

2007-01-01

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M (r) = 52 kDa) and a small (M (r) = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe-S clusters, three [2Fe-2S] clusters and five [4Fe-4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe-S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H(2)-dependent reduction of NAD(+). Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni-Fe active site and a [4Fe-4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni(a)-C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.

Crotoxin: Structural Studies, Mechanism of Action and Cloning of its Gene

DTIC Science & Technology

1988-03-01

thirteen amino acids being acidic . Sequencing of the three peptides present in the acidic subunit, two of which are blocked by pyroglutamate ...the sequence determination of both the basic and acidic subunits of crotoxin- The acidic * subunit peptides were d!Tfficult, .sfi~n~e two of-ftflý...fluorescence spectroscopy. Results indicate a large conformational change occurs upon) ccmplex formation between the acidic and basic subunits of all four

Co-generating synthetic parts toward a self-replicating system

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

Li, Jun; Haas, Wilhelm; Jackson, Kirsten

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE, might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when co-expressed in batchmore » format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in co-expression pool varied from ~1.5 to 5-fold compared to the batch mode, with up to ~ 2.4 µM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ~13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. As a result, this work also points to what is still needed to obtain self-replicating synthetic ribosomes in-situ in the PURE system.« less

Co-generating synthetic parts toward a self-replicating system

DOE PAGES

Li, Jun; Haas, Wilhelm; Jackson, Kirsten; ...

2017-03-23

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE, might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when co-expressed in batchmore » format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in co-expression pool varied from ~1.5 to 5-fold compared to the batch mode, with up to ~ 2.4 µM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ~13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. As a result, this work also points to what is still needed to obtain self-replicating synthetic ribosomes in-situ in the PURE system.« less

Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b from Pleurotus ostreatus

PubMed Central

Ferraroni, Marta; Scozzafava, Andrea; Ullah, Sana; Tron, Thierry; Piscitelli, Alessandra; Sannia, Giovanni

2014-01-01

Laccases are multicopper oxidases of great biotechnological potential. While laccases are generally monomeric glycoproteins, the white-rot fungus Pleurotus ostreatus produces two closely related heterodimeric isoenzymes composed of a large subunit, homologous to the other fungal laccases, and a small subunit. The sequence of the small subunit does not show significant homology to any other protein or domain of known function and consequently its function is unknown. The highest similarity to proteins of known structure is to a putative enoyl-CoA hydratase/isomerase from Acinetobacter baumannii, which shows an identity of 27.8%. Diffraction-quality crystals of the small subunit of the heterodimeric laccase POXA3b (sPOXA3b) from P. ostreatus were obtained using the sitting-drop vapour-diffusion method at 294 K from a solution consisting of 1.8 M sodium formate, 0.1 M Tris–HCl pH 8.5. The crystals belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = 126.6, c = 53.9 Å. The asymmetric unit contains two molecules related by a noncrystallographic twofold axis. A complete data set extending to a maximum resolution of 2.5 Å was collected at 100 K using a wavelength of 1.140 Å. PMID:24419623

Description of Kuraishia piskuri f.a., sp. nov., a new methanol assimilating yeast and transfer of phylogenetically related Candida species to the genera Kuraishia and Nakazawaea as new combinations.

PubMed

Kurtzman, Cletus P; Robnett, Christie J

2014-11-01

The new anamorphic yeast Kuraishia piskuri, f.a., sp. nov. is described for three strains that were isolated from insect frass from trees growing in Florida, USA (type strain, NRRL YB-2544, CBS 13714). Species placement was based on phylogenetic analysis of nuclear gene sequences for the D1/D2 domains of large subunit rRNA, small subunit rRNA, translation elongation factor-1α, and subunits B1 and B2 of RNA polymerase II B. From this analysis, the anamorphic species Candida borneana, Candida cidri, Candida floccosa, Candida hungarica, and Candida ogatae were transferred to the genus Kuraishia as new combinations and Candida anatomiae, Candida ernobii, Candida ishiwadae, Candida laoshanensis, Candida molendini-olei, Candida peltata, Candida pomicola, Candida populi, Candida wickerhamii, and Candida wyomingensis were transferred to the genus Nakazawaea. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation

PubMed Central

Choi, Eunsil; Kang, Nalae; Jeon, Young; Pai, Hyun-Sook

2016-01-01

ABSTRACT The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg2+ concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der-null phenotype. Only K. pneumoniae and S. Typhimurium Der proteins enabled the E. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S. Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. IMPORTANCE In Escherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly in E. coli. Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation in E. coli der mutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits. PMID:27297882

A Puzzle Assembly Strategy for Fabrication of Large Engineered Cartilage Tissue Constructs

PubMed Central

Nover, Adam B.; Jones, Brian K.; Yu, William T.; Donovan, Daniel S.; Podolnick, Jeremy D.; Cook, James L.; Ateshian, Gerard A.; Hung, Clark T.

2016-01-01

Engineering of large articular cartilage tissue constructs remains a challenge as tissue growth is limited by nutrient diffusion. Here, a novel strategy is investigated, generating large constructs through the assembly of individually cultured, interlocking, smaller puzzle-shaped subunits. These constructs can be engineered consistently with more desirable mechanical and biochemical properties than larger constructs (~4-fold greater Young's modulus). A failure testing technique was developed to evaluate the physiologic functionality of constructs, which were cultured as individual subunits for 28 days, then assembled and cultured for an additional 21-35 days. Assembled puzzle constructs withstood large deformations (40-50% compressive strain) prior to failure. Their ability to withstand physiologic loads may be enhanced by increases in subunit strength and assembled culture time. A nude mouse model was utilized to show biocompatibility and fusion of assembled puzzle pieces in vivo. Overall, the technique offers a novel, effective approach to scaling up engineered tissues and may be combined with other techniques and/or applied to the engineering of other tissues. Future studies will aim to optimize this system in an effort to engineer and integrate robust subunits to fill large defects. PMID:26895780

Effects of transgene-encoded high-molecular weight glutenin proteins in wheat flour blends and sponge and dough baking

USDA-ARS?s Scientific Manuscript database

HMW glutenin subunits are the most important determinants of wheat (Triticum aestivum L.) bread-making quality, and subunit composition explains a large percentage of the variability observed between genotypes. Experiments were designed to elevate expression of a key native HMW glutenin subunit (1D...

Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

PubMed Central

Llácer, Jose L.; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R.; Hinnebusch, Alan G.; Ramakrishnan, V.

2015-01-01

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition. PMID:26212456

Large conductance Ca(2+)-activated K(+) channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects.

PubMed

Kirby, R W; Martelli, A; Calderone, V; McKay, N G; Lawson, K

2013-07-15

Large conductance calcium activated potassium channels (BKCa) are fundamental in the control of cellular excitability. Thus, compounds that activate BKCa channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BKCa channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BKCa channel α-subunit alone or α+β1-subunit complex. Channel activity was determined using a non-radioactive Rb(+) efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb(+) efflux both in cells expressing α-subunit alone or α+β1-subunits. Co-expression of the β1-subunit modified the Rb(+) efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC40 values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BKCa channel α+β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC40 values when tested on the BKCa α+β1-subunit expressing cells compared to BKCa α-subunit expressing cells. Further, the Emax values for BKPr4, BKVV and BKH1 were lower in the BKCa channel α+β1-subunit expressing cells. In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BKCa channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Improved purification of brine-shrimp (Artemia saline) (Na+ + K+)-activated adenosine triphosphatase and amino-acid and carbohydrate analyses of the isolated subunits.

PubMed Central

Peterson, G L; Hokin, L E

1980-01-01

Purification of the (Na+ + K+)-activated ATPase has been improved 2-fold the respect to both purity and yield over the previous method [Peterson, Ewing, Hootman & Conte (1978) J. Biol. Chem. 253, 4762-4770] by using Lubrol WX and non-denaturing concentrations of sodium dodecyl sulphate (SDS). The enzyme was purified 200-fold over the homogenate. The preparation had a specific activity of about 600 mumol of Pi/h per mg of protein, and was about 60% pure according to quantification of Coomassie Blue-stained SDS/polyacrylamide gels. The yield of purified enzyme was about 10 mg of protein per 100g of dry brine-shrimp (Artemia salina) cysts. The method is highly suitable for purification either on a small scale (10-25g of dry cysts) or on a large scale (900g of dry cysts) and methods are described for both. The large (Na+ + K+)-activated ATPase subunit (alpha-subunit) was isolated in pure form by SDS-gel filtration on Bio-Gel A 1.5m. The small subunit (beta-subunit) was eluted with other contaminating proteins on the Bio-Gel column, but was isolated in pure form by extraction from SDS/polyacrylamide gels. The amino acid and carbohydrate compositions of both subunits are reported. The alpha-subunit contained 5.2% carbohydrate by weight, and the beta-subunit 9.2%. Sialic acid was absent from both subunits. Images Fig. 3. Fig. 4. PMID:6272692

Chromosome localization of human genes for clathrin adaptor polypeptides AP2{beta} and AP50 and the clathrin-binding protein, VCP

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

Druck, T.; Gu, Y.; Prabhala, G.

1995-11-01

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed {open_quotes}assembly proteins,{close_quotes} or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2{alpha} and AP2{beta}, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2{beta} (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with A1`s. Chromosomal in situ hybridization of a human genomic clonemore » demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2{beta} fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. 13 refs., 2 figs.« less

Modulation of connexin expression and gap junction communication in astrocytes by the gram-positive bacterium S. aureus.

PubMed

Esen, Nilufer; Shuffield, Debbie; Syed, Mohsin M D; Kielian, Tammy

2007-01-01

Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits. Copyright 2006 Wiley-Liss, Inc.

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance.

PubMed

Pausch, Patrick; Müller-Esparza, Hanna; Gleditzsch, Daniel; Altegoer, Florian; Randau, Lennart; Bange, Gert

2017-08-17

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception. Copyright © 2017 Elsevier Inc. All rights reserved.

Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

PubMed

Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

2011-02-01

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta[W

PubMed Central

Orlova, Irina; Nagegowda, Dinesh A.; Kish, Christine M.; Gutensohn, Michael; Maeda, Hiroshi; Varbanova, Marina; Fridman, Eyal; Yamaguchi, Shinjiro; Hanada, Atsushi; Kamiya, Yuji; Krichevsky, Alexander; Citovsky, Vitaly; Pichersky, Eran; Dudareva, Natalia

2009-01-01

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta. PMID:20028839

Inhibition of eukaryotic translation elongation by the antitumor natural product Mycalamide B.

PubMed

Dang, Yongjun; Schneider-Poetsch, Tilman; Eyler, Daniel E; Jewett, John C; Bhat, Shridhar; Rawal, Viresh H; Green, Rachel; Liu, Jun O

2011-08-01

Mycalamide B (MycB) is a marine sponge-derived natural product with potent antitumor activity. Although it has been shown to inhibit protein synthesis, the molecular mechanism of action by MycB remains incompletely understood. We verified the inhibition of translation elongation by in vitro HCV IRES dual luciferase assays, ribosome assembly, and in vivo [(35)S]methinione labeling experiments. Similar to cycloheximide (CHX), MycB inhibits translation elongation through blockade of eEF2-mediated translocation without affecting the eEF1A-mediated loading of tRNA onto the ribosome, AUG recognition, or dipeptide synthesis. Using chemical footprinting, we identified the MycB binding site proximal to the C3993 28S rRNA residue on the large ribosomal subunit. However, there are also subtle, but significant differences in the detailed mechanisms of action of MycB and CHX. First, MycB arrests the ribosome on the mRNA one codon ahead of CHX. Second, MycB specifically blocked tRNA binding to the E-site of the large ribosomal subunit. Moreover, they display different polysome profiles in vivo. Together, these observations shed new light on the mechanism of inhibition of translation elongation by MycB.

Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity.

PubMed

Laterre, Raphaëlle; Pottier, Mathieu; Remacle, Claire; Boutry, Marc

2017-04-01

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO 2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco ( Nicotiana tabacum ) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher V max and K m values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO 2 is released by the active specialized metabolism. © 2017 American Society of Plant Biologists. All Rights Reserved.

A Proteasome Cap Subunit Required for Spindle Pole Body Duplication in Yeast

PubMed Central

McDonald, Heather B.; Byers, Breck

1997-01-01

Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression. The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core. We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S. cerevisiae SUG1/ CIM3 and CIM5 products. This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G. Sathyanarayana, and S.A. Johnston. 1996. J. Biol. Chem. 271:32810– 32817). We have shown that PCS1 function is essential for viability. A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA. EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants. Additionally, we have shown localization of a functional Pcs1–green fluorescent protein fusion to the nucleus throughout the cell cycle. We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication. PMID:9151663

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

PubMed

Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F; Larson, Victoria S; Valasek, Leos; Hinnebusch, Alan G; Lorsch, Jon R

2013-02-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

ICP22 and the UL13 Protein Kinase Are both Required for Herpes Simplex Virus-Induced Modification of the Large Subunit of RNA Polymerase II

PubMed Central

Long, Melissa C.; Leong, Vivian; Schaffer, Priscilla A.; Spencer, Charlotte A.; Rice, Stephen A.

1999-01-01

Herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the large subunit of RNA polymerase II (RNAP II), resulting in the depletion of the hypophosphorylated and hyperphosphorylated forms of this polypeptide (known as IIa and IIo, respectively) and induction of a novel, alternatively phosphorylated form (designated IIi). We previously showed that the HSV-1 immediate-early protein ICP22 is involved in this phenomenon, since induction of IIi and depletion of IIa are deficient in cells infected with 22/n199, an HSV-1 ICP22 nonsense mutant (S. A. Rice, M. C. Long, V. Lam, P. A. Schaffer, and C. A. Spencer, J. Virol. 69:5550–5559, 1995). However, depletion of IIo still occurs in 22/n199-infected cells. This suggests either that another viral gene product affects the RNAP II large subunit or that the truncated ICP22 polypeptide encoded by 22/n199 retains residual activity which leads to IIo depletion. To distinguish between these possibilities, we engineered an HSV-1 ICP22 null mutant, d22-lacZ, and compared it to 22/n199. The two mutants are indistinguishable in their effects on the RNAP II large subunit, suggesting that an additional viral gene product is involved in altering RNAP II. Two candidates are UL13, a protein kinase which has been implicated in ICP22 phosphorylation, and the virion host shutoff (Vhs) factor, the expression of which is positively regulated by ICP22 and UL13. To test whether UL13 is involved, a UL13-deficient viral mutant, d13-lacZ, was engineered. This mutant was defective in IIi induction and IIa depletion, displaying a phenotype very similar to that of d22-lacZ. In contrast, a Vhs mutant had effects that were indistinguishable from wild-type HSV-1. Therefore, UL13 but not the Vhs function plays a role in modifying the RNAP II large subunit. To study the potential role of UL13 in viral transcription, we carried out nuclear run-on transcription analyses in infected human embryonic lung cells. Infections with either UL13 or ICP22 mutants led to significantly reduced amounts of viral genome transcription at late times after infection. Together, our results suggest that ICP22 and UL13 are involved in a common pathway that alters RNAP II phosphorylation and that in some cell lines this change promotes viral late transcription. PMID:10364308

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors

PubMed Central

Nguyen, Phuong T.; Yarov-Yarovoy, Vladimir; Burns, Marie E.; Pugh, Edward N.

2017-01-01

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors’ subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gαt), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gαt the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s−1. Analyzing swelling as osmotically driven water influx, we find the H2O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10−5 cm⋅s−1, comparable to that of other cells lacking aquaporin expression. Application of Van’t Hoff’s law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H2O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity. PMID:28320964

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, Robert L.

1998-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, R.L.

1998-03-03

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, R.L.

1999-02-02

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits.

PubMed

Fernández-Pevida, Antonio; Martín-Villanueva, Sara; Murat, Guillaume; Lacombe, Thierry; Kressler, Dieter; de la Cruz, Jesús

2016-09-19

The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hexadecameric structure of an invertebrate gap junction channel.

PubMed

Oshima, Atsunori; Matsuzawa, Tomohiro; Murata, Kazuyoshi; Tani, Kazutoshi; Fujiyoshi, Yoshinori

2016-03-27

Innexins are invertebrate-specific gap junction proteins with four transmembrane helices. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of connexin gap junction channels, few structural studies of recombinant innexin channels are reported. Here we show the three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. The N-terminal deleted INX-6 proteins are crystallized in lipid bilayers. The three-dimensional reconstruction determined by cryo-electron crystallography reveals that a single INX-6 gap junction channel comprises 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which comprise 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26. Two bulb densities are observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. These findings imply a structural diversity of gap junction channels among multicellular organisms. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg²⁺ binding to the MIDAS.

PubMed

Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J; Filizola, Marta; Springer, Timothy A; Coller, Barry S

2012-03-14

An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

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

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface inmore » which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.« less

The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

PubMed Central

Fernández-Pevida, Antonio; Martín-Villanueva, Sara; Murat, Guillaume; Lacombe, Thierry; Kressler, Dieter; de la Cruz, Jesús

2016-01-01

The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D. PMID:27422873

The yeast genome may harbor hypoxia response elements (HRE).

PubMed

Ferreira, Túlio César; Hertzberg, Libi; Gassmann, Max; Campos, Elida Geralda

2007-01-01

The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated when cells are submitted to hypoxia. The heterodimer is composed of two subunits, HIF-1alpha and the constitutively expressed HIF-1beta. During normoxia, HIF-1alpha is degraded by the 26S proteasome, but hypoxia causes HIF-1alpha to be stabilized, enter the nucleus and bind to HIF-1beta, thus forming the active complex. The complex then binds to the regulatory sequences of various genes involved in physiological and pathological processes. The specific regulatory sequence recognized by HIF-1 is the hypoxia response element (HRE) that has the consensus sequence 5'BRCGTGVBBB3'. Although the basic transcriptional regulation machinery is conserved between yeast and mammals, Saccharomyces cerevisiae does not express HIF-1 subunits. However, we hypothesized that baker's yeast has a protein analogous to HIF-1 which participates in the response to changes in oxygen levels by binding to HRE sequences. In this study we screened the yeast genome for HREs using probabilistic motif search tools. We described 24 yeast genes containing motifs with high probability of being HREs (p-value<0.1) and classified them according to biological function. Our results show that S. cerevisiae may harbor HREs and indicate that a transcription factor analogous to HIF-1 may exist in this organism.

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

PubMed Central

Kiparisov, S.; Sergiev, P. V.; Dontsova, O. A.; Petrov, A.; Meskauskas, A.; Dinman, J. D.

2005-01-01

5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semidominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression. PMID:16047201

The secreted form of the p40 subunit of interleukin (IL)-12 inhibits IL-23 functions and abrogates IL-23-mediated antitumour effects

PubMed Central

Shimozato, Osamu; Ugai, Shin-ichi; Chiyo, Masako; Takenobu, Hisanori; Nagakawa, Hiroyasu; Wada, Akihiko; Kawamura, Kiyoko; Yamamoto, Hiroshi; Tagawa, Masatoshi

2006-01-01

Interleukin (IL)-23 is a heterodimeric cytokine consisting of a novel p19 molecule and the p40 subunit of IL-12. Since secreted p40 can act as an antagonist for IL-12, we investigated whether p40 also inhibited IL-23-mediated immunological functions. p40 did not induce interferon (IFN)-γ or IL-17 production from splenocytes but impaired IL-23-induced cytokine production by competitive binding to the IL-23 receptors. Furthermore, a mixed population of murine colon carcinoma Colon 26 cells transduced with the p40 gene and those transduced with the IL-23 gene developed tumours in syngenic mice, whereas the IL-23-expressing Colon 26 cells were completely rejected. p40 also suppressed IFN-γ production of antigen-stimulated splenocytes and IL-23-mediated cytotoxic T-lymphocyte activities in the mice that rejected Colon 26 cells expressing IL-23. p40 can thereby antagonize IL-23 and is a possible therapeutic agent for suppression of IL-23 functions. PMID:16423037

Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

PubMed

Thabet, Rahma; Rouault, J-D; Ayadi, Habib; Leignel, Vincent

2016-01-01

The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Topology of subunits of the mammalian cytochrome c oxidase: Relationship to the assembly of the enzyme complex

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

Yu-Zhong Zhang; Ewart, G.; Capaldi, R.A.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane({sup 35}S)sulfonate and sodium methyl 4-({sup 3}H)formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibodymore » (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C-domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage produce from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.« less

An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

PubMed

Shiota, Masaki; Yamazaki, Tomohiko; Yoshimatsu, Keiichi; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2016-12-01

Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification and characterization of hemagglutinating proteins from Poker-chip Venus (Meretrix lusoria) and Corbicula clam (Corbicula fluminea).

PubMed

Cheng, Chin-Fu; Hung, Shao-Wen; Chang, Yung-Chung; Chen, Ming-Hui; Chang, Chen-Hsuan; Tsou, Li-Tse; Tu, Ching-Yu; Lin, Yu-Hsing; Liu, Pan-Chen; Lin, Shiun-Long; Wang, Way-Shyan

2012-01-01

Hemagglutinating proteins (HAPs) were purified from Poker-chip Venus (Meretrix lusoria) and Corbicula clam (Corbicula fluminea) using gel-filtration chromatography on a Sephacryl S-300 column. The molecular weights of the HAPs obtained from Poker-chip Venus and Corbicula clam were 358 kDa and 380 kDa, respectively. Purified HAP from Poker-chip Venus yielded two subunits with molecular weights of 26 kDa and 29 kDa. However, only one HAP subunit was purified from Corbicula clam, and its molecular weight was 32 kDa. The two Poker-chip Venus HAPs possessed hemagglutinating ability (HAA) for erythrocytes of some vertebrate animal species, especially tilapia. Moreover, HAA of the HAP purified from Poker-chip Venus was higher than that of the HAP of Corbicula clam. Furthermore, Poker-chip Venus HAPs possessed better HAA at a pH higher than 7.0. When the temperature was at 4°C-10°C or the salinity was less than 0.5‰, the two Poker-chip Venus HAPs possessed better HAA compared with that of Corbicula clam.

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.

PubMed

Chojnacki, Michal; Mansour, Wissam; Hameed, Dharjath S; Singh, Rajesh K; El Oualid, Farid; Rosenzweig, Rina; Nakasone, Mark A; Yu, Zanlin; Glaser, Fabian; Kay, Lewis E; Fushman, David; Ovaa, Huib; Glickman, Michael H

2017-04-20

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub PT ), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub PT , and we expect that its future uses will help define and investigate the ubiquitin interactome. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Spreitzer, Robert J.

CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesismore » is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.« less

ApoE4 expression accelerates hippocampus-dependent cognitive deficits by enhancing Aβ impairment of insulin signaling in an Alzheimer’s disease mouse model

PubMed Central

Chan, Elizabeth S.; Shetty, Mahesh Shivarama; Sajikumar, Sreedharan; Chen, Christopher; Soong, Tuck Wah; Wong, Boon-Seng

2016-01-01

The apolipoprotein E4 (ApoE4) is the strongest genetic risk factor for Alzheimer’s disease (AD). The AD brain was shown to be insulin resistant at end stage, but the interplay between insulin signaling, ApoE4 and Aβ across time, and their involvement in memory decline is unclear. To investigate insulin response in the ageing mouse hippocampus, we crossed the human ApoE-targeted replacement mice with the mutant human amyloid precursor protein (APP) mice (ApoExAPP). While hippocampal Aβ levels were comparable between ApoE3xAPP and ApoE4xAPP mice at 26 weeks, insulin response was impaired in the ApoE4xAPP hippocampus. Insulin treatment was only able to stimulate insulin signaling and increased AMPA-GluR1 phosphorylation in forskolin pre-treated hippocampal slices from ApoE3xAPP mice. In ApoE4xAPP mice, insulin dysfunction was also associated with poorer spatial memory performance. Using dissociated hippocampal neuron in vitro, we showed that insulin response in ApoE3 and ApoE4 neurons increased AMPA receptor-mediated miniature excitatory postsynaptic current (mEPSC) amplitudes and GluR1-subunit insertion. Pre-treatment of ApoE3 neurons with Aβ42 did not affect insulin-mediated GluR1 subunit insertion. However, impaired insulin sensitivity observed only in the presence of ApoE4 and Aβ42, attenuated GluR1-subunit insertion. Taken together, our results suggest that ApoE4 enhances Aβ inhibition of insulin-stimulated AMPA receptor function, which accelerates memory impairment in ApoE4xAPP mice. PMID:27189808

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

PubMed Central

Ma, Long; Tan, Zhiping; Teng, Yanling; Hoersch, Sebastian; Horvitz, H. Robert

2011-01-01

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3′ splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3′ splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3′ splice sites. PMID:22033331

Retromer association with membranes: plants have their own rules!

PubMed

Zelazny, Enric; Santambrogio, Martina; Gaude, Thierry

2013-09-01

The retromer is an endosome-localized complex involved in protein trafficking. To better understand its function and regulation in plants, we recently investigated how Arabidopsis retromer subunits assemble and are targeted to endosomal membranes and highlighted original features compared with mammals. We characterized Arabidopsis vps26 null mutant and showed that it displays severe developmental defaults similar to those observed in vps29 mutant. Here, we go further by describing new phenotypic defects associated with loss of VPS26 function, such as inhibition of lateral root initiation. Recently, we showed that VPS35 subunit plays a crucial role in the recruitment of the plant retromer to endosomes, probably through an interaction with the Rab7 homolog RABG3f. In this work, we now show that contrary to mammals, Arabidopsis Rab5 homologs do not seem to be necessary for the recruitment of the core retromer to endosomal membranes, which highlights a new specificity of the plant retromer.

Genetic diversity and classification of Tibetan yak populations based on the mtDNA COIII gene.

PubMed

Song, Q Q; Chai, Z X; Xin, J W; Zhao, S J; Ji, Q M; Zhang, C F; Ma, Z J; Zhong, J C

2015-03-13

To determine the level of genetic diversity and phylogenetic relationships among Tibetan yak populations, the mitochondrial DNA cytochrome c oxidase subunit 3 (COIII) genes of 378 yak individuals from 16 populations were analyzed in this study. The results showed that the length of cytochrome c oxidase subunit 3 gene sequences was 781 bp, with nucleotide frequencies of 29.2, 29.4, 26.1, and 15.2% for T, C, A, and G, respectively. A total of 26 haplotypes were identified, with 69 polymorphic sites, including 11 parsimony-informative sites and 58 single-nucleotide polymorphism sites. No deletions/insertions were found in sequence comparison, indicating that nucleotide mutation types were transitions and transversions. Haplotype and nucleotide diversities were 0.562 and 0.00138, respectively, indicating a high level of genetic diversity in Tibetan yak populations. Phylogenetic relationship analysis indicated that Tibetan yak populations are divided into 2 groups.

The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes.

PubMed

Schäfer, Thorsten; Strauss, Daniela; Petfalski, Elisabeth; Tollervey, David; Hurt, Ed

2003-03-17

Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood. Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes. During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (approximately 30 species) dissociate, and significantly fewer factors associate with 40S pre-ribosomes. Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g. Enp1p, Dim1p and Rrp12p), whereas others (e.g. Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits. Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits. Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm. This pathway involves fewer changes in composition than seen during 60S biogenesis.

Directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S13 using tethered Fe(II).

PubMed Central

Heilek, G M; Noller, H F

1996-01-01

Directed hydroxyl radical probing was used to probe the rRNA neighborhood around protein S13 in the 30S ribosomal subunit. The unique cysteine at position 84 of S13 served as a tethering site for attachment of Fe(II)-1-(p-bromoacetamidobenzyl)-EDTA. Derivatized S13 (Fe-C84-S13) was then assembled into 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe(II) resulted in cleavage of the RNA backbone in two localized regions of the 3' major domain of 16S rRNA. One region spans nt 1308-1333 and is close to a site previously crosslinked to S13. A second set of cleavages is found in the 950/1230 helix. Both regions have been implicated in binding of S13 by previous chemical footprinting studies using base-specific chemical probes and solution-based hydroxyl radical probing. These results place both regions of 16S rRNA in proximity to position C84 of S13 in the three-dimensional structure of the 30S ribosomal subunit. PMID:8718688

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

The speEspeD operon of Escherichia coli. Formation and processing of a proenzyme form of S-adenosylmethionine decarboxylase.

PubMed

Tabor, C W; Tabor, H

1987-11-25

We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C. W., Tabor, H., and Xie, Q.-W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6040-6044). We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme. Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit. The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity. Both subunits are present in the purified enzyme. These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D. L., and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822).

β-hydroxy-β-methylbutyrate (HMB) Prevents Sepsis-Induced Diaphragm Dysfunction in Mice

PubMed Central

Supinski, Gerald S.; Callahan, Leigh Ann

2014-01-01

Infections induce severe respiratory muscle weakness. Currently there are no treatments for this important clinical problem. We tested the hypothesis that β-hydroxy-β-methylbutyrate (HMB) would prevent sepsis-induced diaphragm weakness. Four groups of adult male mice were studied: controls (saline-injected), sepsis (intraperitoneal lipopolysaccharide), sepsis+HMB (injected intravenously), and HMB. Diaphragm force generation and indices of caspase 3, calpain, 20S proteasomal subunit, and double-stranded RNA-dependent protein kinase (PKR) activation were assessed after 24 hours. Sepsis elicited large reductions in diaphragm specific force generation at all stimulation frequencies. Endotoxin also activated caspase 3, calpain, the 20S proteasomal subunit and PKR in the diaphragm. HMB blocked sepsis-induced caspase 3, 20S proteasomal and PKR activation, but did not prevent calpain activation. Most importantly, HMB administration significantly attenuated sepsis-induced diaphragm weakness, preserving muscle force generation at all stimulation frequencies (p<0.01). We speculate that HMB may prove to be an important therapy in infected patients, with the potential to increase diaphragm strength, to reduce the duration of mechanical ventilation and to decrease mortality in this patient population PMID:24632527

Interaction of tetracycline with RNA: photoincorporation into ribosomal RNA of Escherichia coli.

PubMed Central

Oehler, R; Polacek, N; Steiner, G; Barta, A

1997-01-01

Photolysis of [3H]tetracycline in the presence of Escherichia coli ribosomes results in an approximately 1:1 ratio of labelling ribosomal proteins and RNAs. In this work we characterize crosslinks to both 16S and 23S RNAs. Previously, the main target of photoincorporation of [3H]tetracycline into ribosomal proteins was shown to be S7, which is also part of the one strong binding site of tetracycline on the 30S subunit. The crosslinks on 23S RNA map exclusively to the central loop of domain V (G2505, G2576 and G2608) which is part of the peptidyl transferase region. However, experiments performed with chimeric ribosomal subunits demonstrate that peptidyltransferase activity is not affected by tetracycline crosslinked solely to the 50S subunits. Three different positions are labelled on the 16S RNA, G693, G1300 and G1338. The positions of these crosslinked nucleotides correlate well with footprints on the 16S RNA produced either by tRNA or the protein S7. This suggests that the nucleotides are labelled by tetracycline bound to the strong binding site on the 30S subunit. In addition, our results demonstrate that the well known inhibition of tRNA binding to the A-site is solely due to tetracycline crosslinked to 30S subunits and furthermore suggest that interactions of the antibiotic with 16S RNA might be involved in its mode of action. PMID:9092632

Identification and properties of the largest subunit of the DNA-dependent RNA polymerase of fish lymphocystis disease virus: dramatic difference in the domain organization in the family Iridoviridae.

PubMed

Müller, M; Schnitzler, P; Koonin, E V; Darai, G

1995-05-01

Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of the known largest subunits of DdRPs from different species contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains (RQP[T/S]LH and NADFDGDE) were used for detecting the corresponding gene of fish lymphocystis disease virus (FLCDV), a member of the family Iridoviridae, which replicates in the cytoplasm of infected cells of flatfish. The gene coding for the largest subunit of the DdRP was identified using a PCR-derived probe. The screening of the complete EcoRI gene library of the viral genome led to the identification of the gene locus of the largest subunit of the DdRP within the EcoRI DNA fragment B (12.4 kbp, 0.034 to 0.165 map units). The nucleotide sequence of a part (8334 bp) of the EcoRI DNA fragment B was determined and a large ORF on the lower strand (ATG = 5787; TAA = 2190) was detected which encodes a protein of 1199 amino acids. Comparison of the amino acid sequences of the largest subunits of the DdRP (RPO1) of FLCDV and Chilo iridescent virus (CIV) revealed a dramatic difference in their domain organization. Unlike the 1051 aa RPO1 of CIV, which lacks the C-terminal domain conserved in eukaryotic, eubacterial and other viral RNA polymerases, the 1199 aa RPO1 of FLCDV is fully collinear with its cellular and viral homologues. Despite this difference, comparative analysis of the amino acid sequences of viral and cellular RNA polymerases suggests a common origin for the largest RNA polymerase subunits of FLCDV and CIV.

Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the beta'-subunit of RNA polymerase.

PubMed Central

Cozens, A L; Walker, J E

1986-01-01

The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome. It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex. The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes. It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E. coli RNA polymerase and to yeast RNA polymerases II and III. PMID:3530249

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor

PubMed Central

Simonetti, Angelita; Marzi, Stefano; Billas, Isabelle M. L.; Tsai, Albert; Fabbretti, Attilio; Myasnikov, Alexander G.; Roblin, Pierre; Vaiana, Andrea C.; Hazemann, Isabelle; Eiler, Daniel; Steitz, Thomas A.; Puglisi, Joseph D.; Gualerzi, Claudio O.; Klaholz, Bruno P.

2013-01-01

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNAfMet positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process. PMID:24029017

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae

PubMed Central

Hierlmeier, Thomas; Merl, Juliane; Sauert, Martina; Perez-Fernandez, Jorge; Schultz, Patrick; Bruckmann, Astrid; Hamperl, Stephan; Ohmayer, Uli; Rachel, Reinhard; Jacob, Anja; Hergert, Kristin; Deutzmann, Rainer; Griesenbeck, Joachim; Hurt, Ed; Milkereit, Philipp; Baßler, Jochen; Tschochner, Herbert

2013-01-01

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p–Noc1p–Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity. PMID:23209026

A ruby-colored Pseudobaeospora species is described as new from material collected on the island of Hawaii.

PubMed

Desjardin, Dennis E; Hemmes, Don E; Perry, Brian A

2014-01-01

Pseudobaeospora wipapatiae is described as new based on material collected in alien wet habitats on the island of Hawaii. Unique features of this beautiful species include deep ruby-colored basidiomes with two-spored basidia, amyloid cheilocystidia and a hymeniderm pileipellis with abundant pileocystidia that is initially deep ruby in KOH then changes to lilac gray. Phylogenetic analysis of nuclear large ribosomal subunit sequence data suggest a close relationship between Pseudobaeospora and Tricholoma. BLAST comparisons of internal transcribed spacer and 5.8S nuclear ribosomal subunit regions sequence data reveal greatest similarity with existing sequences of Pseudobaeospora species. A comprehensive description, color photograph, illustrations of salient micromorphological features and comparisons with phenetically similar taxa are provided. © 2014 by The Mycological Society of America.

S-nitrosylation of the IGF-1 receptor disrupts the cell proliferative action of IGF-1.

PubMed

Okada, Kazushi; Zhu, Bao-Ting

2017-09-30

The insulin-like growth factor 1 receptor (IGF-1R) is a disulfide-linked heterotetramer containing two α-subunits and two β-subunits. Earlier studies demonstrate that nitric oxide (NO) can adversely affect IGF-1 action in the central nervous system. It is known that NO can induce S-nitrosylation of the cysteine residues in proteins, thereby partly contributing to the regulation of protein function. In the present study, we sought to determine whether S-nitrosylation of the cysteine residues in IGF-1R is an important post-translational modification that regulates its response to IGF-1. Using cultured SH-SY5Y human neuroblastoma cells as an in vitro model, we found that treatment of cells with S-nitroso-cysteine (SNOC), a NO donor that can nitrosylate the cysteine residues in proteins, induces S-nitrosylation of the β subunit of IGF-1R but not its α-subunit. IGF-1Rβ S-nitrosylation by SNOC is coupled with increased dissociation of the IGF-1R protein complex. In addition, disruption of the IGF-1R function resulting from S-nitrosylation of the IGF-1Rβ subunit is associated with disruption of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Further, we observed that SNOC-induced IGF-1Rβ S-nitrosylation results in a dose-dependent inhibition of cell proliferation and survival. Together, these results suggest that elevated nitrosative stress may result in dysfunction of cellular IGF-1R signaling through S-nitrosylation of the cysteine residues in the IGF-1Rβ subunit, thereby disrupting the downstream PI3K and MAPK signaling functions and ultimately resulting in inhibition of cell proliferation and survival. Copyright © 2017. Published by Elsevier Inc.

Revisiting the phylogeny of Ocellularieae, the second largest tribe within Graphidaceae (lichenized Ascomycota: Ostropales)

Treesearch

Ekaphan Kraichak; Sittiporn Parnmen; Robert Lücking; Eimy Rivas Plata; Andre Aptroot; Marcela E.S. Caceres; Damien Ertz; Armin Mangold; Joel A. Mercado-Diaz; Khwanruan Papong; Dries Van der Broeck; Gothamie Weerakoon; H. Thorsten Lumbsch; NO-VALUE

2014-01-01

We present an updated 3-locus molecular phylogeny of tribe Ocellularieae, the second largest tribe within subfamily Graphidoideae in the Graphidaceae. Adding 165 newly generated sequences from the mitochondrial small subunit rDNA (mtSSU), the nuclear large subunit rDNA (nuLSU), and the second largest subunit of the DNA-directed RNA polymerase II (RPB2), we currently...

Gene Cloning and Characterization of the Very Large NAD-Dependent l-Glutamate Dehydrogenase from the Psychrophile Janthinobacterium lividum, Isolated from Cold Soil▿

PubMed Central

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-01-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids. PMID:17526698

Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-08-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.

Forskolin-mediated BeWo cell fusion involves down-regulation of miR-92a-1-5p that targets dysferlin and protein kinase cAMP-activated catalytic subunit alpha.

PubMed

Dubey, Richa; Malhotra, Sudha S; Gupta, Satish K

2018-06-01

To study the role of miRNA(s) during trophoblastic BeWo cell fusion. Changes in miRNA(s) profile of BeWo cells treated with forskolin were analyzed using Affymetrix miRNA microarray platform. Down-regulated miRNA, miR-92a-1-5p, was overexpressed in BeWo cells followed by forskolin treatment to understand its relevance in the process of BeWo cell fusion by desmoplakin I+II staining and hCG secretion by ELISA. Predicted targets of miR-92a-1-5p were also confirmed by qRT-PCR/Western blotting. The miRNA profiling of BeWo cells after forskolin (25 μmol/L) treatment identified miR-92a-1-5p as the most significantly down-regulated miRNA both at 24 and 48 hours time points. Overexpression of miR-92a-1-5p in these cells led to a significant decrease in forskolin-mediated cell fusion and hCG secretion. miRNA target prediction software, TargetScan, revealed dysferlin (DYSF) and protein kinase cAMP-activated catalytic subunit alpha (PRKACA), as target genes of miR-92a-1-5p. Overexpression of miR-92a-1-5p in BeWo cells showed reduction in forskolin-induced transcripts for DYSF and PRKACA. Further, reduction in DYSF (~2.6-fold) at protein level and PRKACA-encoded protein kinase A catalytic subunit alpha (PKAC-α; ~1.6-fold) were also observed. These observations suggest that miR-92a-1-5p regulates forskolin-mediated BeWo cell fusion and hCG secretion by regulating PKA signaling pathway and dysferlin expression. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biology of Symbioses between Marine Invertebrates and Intracellular Bacteria

DTIC Science & Technology

1990-01-30

bisphosphate carboxylase We designed from published sequence information oligonucleotide primers which are complementary to conserved regions on RubisCO ...large and small subunit genes. These primers were used successfully to amplify using polymerase chain reaction (PCR) specific regions of RubisCO ...for the large subunit of ribulose bisphosphate carboxylase/oxygenase ( RubisCO ) to symbiont DNA shows that the symbionts from both deep-sea and shallow

Cloning and sequencing of the allophycocyanin genes from Spirulina maxima (Cyanophyta)

NASA Astrophysics Data System (ADS)

Qin, Song; Hiroyuki, Kojima; Yoshikazu, Kawata; Shin-Ichi, Yano; Zeng, Cheng-Kui

1998-03-01

The genes coding for the α-and β-subunit of allophycocyanin ( apcA and apcB) from the cyanophyte Spirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotide sequence similarity and 30.4% of similarity of deduced amino acid sequence between them. The amino acid sequence identities between S. maxima and S. platensis are 99.4% for α subunit and 100% for β subunit.

Systematic relationships among Lutzomyia sand flies (Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA sequences.

PubMed

Beati, Lorenza; Cáceres, Abraham G; Lee, Jamie A; Munstermann, Leonard E

2004-02-01

Lutzomyia spp. are New World phlebotomine sand flies, many of which are involved in the transmission of human diseases, such as leishmaniases and bartonellosis. The systematic classification of the approximately 400 species in the genus has been based on morphological characters, but the relationships within the genus are still very much in question. We have inferred phylogenies of 32 species of phlebotomine sand flies belonging to seven sub-genera and two species groups, by using fragments of the mitochondrial small subunit (12SrRNA) and of the nuclear large subunit (28SrRNA) ribosomal gene sequences. The subgenus Helcocyrtomyia and the Verrucarum species group, prominent representatives of the Peruvian sand fly fauna, were represented by 11 and 7 species, respectively. Although based on a limited number of taxa, the resulting phylogenies, based on 837 characters, provide an initial phylogenetic backbone for the progressive reconstruction of infrageneric relationships within Lutzomyia.

The Structure of the Anti-tuberculosis Antibiotics Viomycin and Capreomycin Bound to the 70S Ribosome

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

Stanley, R.; Blaha, G; Grodzicki, R

2010-01-01

Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-{angstrom} resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in themore » pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.« less

The structures of the anti-tuberculosis antibiotics viomycin and capreomycin bound to the 70S ribosome

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

Stanley, Robin E.; Blaha, Gregor; Grodzicki, Robert L.

2010-05-03

Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-{angstrom} resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in themore » pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.« less

The universally conserved GTPase HflX is an RNA helicase that restores heat-damaged Escherichia coli ribosomes.

PubMed

Dey, Sandip; Biswas, Chiranjit; Sengupta, Jayati

2018-06-21

The ribosome-associated GTPase HflX acts as an antiassociation factor upon binding to the 50S ribosomal subunit during heat stress in Escherichia coli Although HflX is recognized as a guanosine triphosphatase, several studies have shown that the N-terminal domain 1 of HflX is capable of hydrolyzing adenosine triphosphate (ATP), but the functional role of its adenosine triphosphatase (ATPase) activity remains unknown. We demonstrate that E. coli HflX possesses ATP-dependent RNA helicase activity and is capable of unwinding large subunit ribosomal RNA. A cryo-electron microscopy structure of the 50S-HflX complex in the presence of nonhydrolyzable analogues of ATP and guanosine triphosphate hints at a mode of action for the RNA helicase and suggests the linker helical domain may have a determinant role in RNA unwinding. Heat stress results in inactivation of the ribosome, and we show that HflX can restore heat-damaged ribosomes and improve cell survival. © 2018 Dey et al.

Protein and quality analyses of accessions from the USDA soybean germplasm collection for tofu production.

PubMed

Meng, Shi; Chang, Sam; Gillen, Anne M; Zhang, Yan

2016-12-15

Food-grade soybeans with large seed size, uniformity, clear hilum and a high 11S/7S ratio are favoured by the food industry for making tofu. In order to search for soybean lines with desirable characteristics for making foods, 22 soybean lines were selected from the USDA-Soybean Germplasm Collection, were grown in Stoneville, MS for biochemical analysis and tofu texture and sensory quality tests. Eight lines were identified, from 22 lines harvested in 2014, to be suitable for tofu making, as judged by chemical composition and sensory quality of pressed tofu. In the filled tofu making and texture analysis study, the correlation between A3 subunit content and filled tofu firmness was significant (N=22, r=0.77, P<0.001). The results indicated that the A3 subunit could be an indicator for predicting the firmness of tofu. The results provided important food quality information for the selection of soybean genotypes for improving food quality. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular basis for the interaction between Integrator subunits IntS9 and IntS11 and its functional importance.

PubMed

Wu, Yixuan; Albrecht, Todd R; Baillat, David; Wagner, Eric J; Tong, Liang

2017-04-25

The metazoan Integrator complex (INT) has important functions in the 3'-end processing of noncoding RNAs, including the uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA), and in the transcription of coding genes by RNA polymerase II. The INT contains at least 14 subunits, but its molecular mechanism of action is poorly understood, because currently there is little structural information about its subunits. The endonuclease activity of INT is mediated by its subunit 11 (IntS11), which belongs to the metallo-β-lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3'-end processing. IntS11 forms a stable complex with Integrator complex subunit 9 (IntS9) through their C-terminal domains (CTDs). Here, we report the crystal structure of the IntS9-IntS11 CTD complex at 2.1-Å resolution and detailed, structure-based biochemical and functional studies. The complex is composed of a continuous nine-stranded β-sheet with four strands from IntS9 and five from IntS11. Highly conserved residues are located in the extensive interface between the two CTDs. Yeast two-hybrid assays and coimmunoprecipitation experiments confirm the structural observations on the complex. Functional studies demonstrate that the IntS9-IntS11 interaction is crucial for the role of INT in snRNA 3'-end processing.

Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

PubMed Central

Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

2014-01-01

Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits.

PubMed

Torres, Yolima P; Granados, Sara T; Latorre, Ramón

2014-01-01

Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca(2+) concentration, the large conductance voltage- and Ca(2+)-activated K(+) channel (BK) is unique among the superfamily of K(+) channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K(+) channels) and a large C terminus composed of two regulators of K(+) conductance domains (RCK domains), where the Ca(2+)-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca(2+) sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above.

Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

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

Svergun, D.I.; Koch, M.H.J.; Pedersen, J.S.

1994-12-31

The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from themore » protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.« less

Crystallization of the Nonameric Small Terminase Subunit of Bacteriophage P22

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

A Roy; A Bhardwaj; G Cingolani

2011-12-31

The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometrymore » of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.« less

Crystallization of the Nonameric Small Terminase Subunit of bacteriophage P22

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

A Roy; A Bhardwaj; G Cingoloni

2011-12-31

The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometrymore » of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.« less

The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme.

PubMed

Tuncel, Aytug; Kawaguchi, Joe; Ihara, Yasuharu; Matsusaka, Hiroaki; Nishi, Aiko; Nakamura, Tetsuhiro; Kuhara, Satoru; Hirakawa, Hideki; Nakamura, Yasunori; Cakir, Bilal; Nagamine, Ai; Okita, Thomas W; Hwang, Seon-Kap; Satoh, Hikaru

2014-06-01

Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A New Splice Variant of Large Conductance Ca2+-activated K+ (BK) Channel α Subunit Alters Human Chondrocyte Function.

PubMed

Suzuki, Yoshiaki; Ohya, Susumu; Yamamura, Hisao; Giles, Wayne R; Imaizumi, Yuji

2016-11-11

Large conductance Ca 2+ -activated K + (BK) channels play essential roles in both excitable and non-excitable cells. For example, in chondrocytes, agonist-induced Ca 2+ release from intracellular store activates BK channels, and this hyperpolarizes these cells, augments Ca 2+ entry, and forms a positive feed-back mechanism for Ca 2+ signaling and stimulation-secretion coupling. In the present study, functional roles of a newly identified splice variant in the BK channel α subunit (BKαΔe2) were examined in a human chondrocyte cell line, OUMS-27, and in a HEK293 expression system. Although BKαΔe2 lacks exon2, which codes the intracellular S0-S1 linker (Glu-127-Leu-180), significant expression was detected in several tissues from humans and mice. Molecular image analyses revealed that BKαΔe2 channels are not expressed on plasma membrane but can traffic to the plasma membrane after forming hetero-tetramer units with wild-type BKα (BKαWT). Single-channel current analyses demonstrated that BKα hetero-tetramers containing one, two, or three BKαΔe2 subunits are functional. These hetero-tetramers have a smaller single channel conductance and exhibit lower trafficking efficiency than BKαWT homo-tetramers in a stoichiometry-dependent manner. Site-directed mutagenesis of residues in exon2 identified Helix2 and the linker to S1 (Trp-158-Leu-180, particularly Arg-178) as an essential segment for channel function including voltage dependence and trafficking. BKαΔe2 knockdown in OUMS-27 chondrocytes increased BK current density and augmented the responsiveness to histamine assayed as cyclooxygenase-2 gene expression. These findings provide significant new evidence that BKαΔe2 can modulate cellular responses to physiological stimuli in human chondrocyte and contribute under pathophysiological conditions, such as osteoarthritis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

IKs channels open slowly because KCNE1 accessory subunits slow the movement of S4 voltage sensors in KCNQ1 pore-forming subunits

PubMed Central

Ruscic, Katarina J.; Miceli, Francesco; Villalba-Galea, Carlos A.; Dai, Hui; Mishina, Yukiko; Bezanilla, Francisco; Goldstein, Steve A. N.

2013-01-01

Human IKs channels activate slowly with the onset of cardiac action potentials to repolarize the myocardium. IKs channels are composed of KCNQ1 (Q1) pore-forming subunits that carry S4 voltage-sensor segments and KCNE1 (E1) accessory subunits. Together, Q1 and E1 subunits recapitulate the conductive and kinetic properties of IKs. How E1 modulates Q1 has been unclear. Investigators have variously posited that E1 slows the movement of S4 segments, slows opening and closing of the conduction pore, or modifies both aspects of electromechanical coupling. Here, we show that Q1 gating current can be resolved in the absence of E1, but not in its presence, consistent with slowed movement of the voltage sensor. E1 was directly demonstrated to slow S4 movement with a fluorescent probe on the Q1 voltage sensor. Direct correlation of the kinetics of S4 motion and ionic current indicated that slowing of sensor movement by E1 was both necessary and sufficient to determine the slow-activation time course of IKs. PMID:23359697

Amino acid sequence of the human fibronectin receptor

PubMed Central

1987-01-01

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+- binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors. PMID:2958481

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

PubMed Central

Yáñez, R J; Boursnell, M; Nogal, M L; Yuste, L; Viñuela, E

1993-01-01

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication. Images PMID:8506138

Prp43p Is a DEAH-Box Spliceosome Disassembly Factor Essential for Ribosome Biogenesis

PubMed Central

Combs, D. Joshua; Nagel, Roland J.; Ares, Manuel; Stevens, Scott W.

2006-01-01

The known function of the DEXH/D-box protein Prp43p is the removal of the U2, U5, and U6 snRNPs from the postsplicing lariat-intron ribonucleoprotein complex. We demonstrate that affinity-purified Prp43p-associated material includes the expected spliceosomal components; however, we also identify several preribosomal complexes that are specifically purified with Prp43p. Conditional prp43 mutant alleles confer a 35S pre-rRNA processing defect, with subsequent depletion of 27S and 20S precursors. Upon a shift to a nonpermissive temperature, both large and small-ribosomal-subunit proteins accumulate in the nucleolus of prp43 mutants. Pulse-chase analysis demonstrates delayed kinetics of 35S, 27S, and 20S pre-rRNA processing with turnover of these intermediates. Microarray analysis of pre-mRNA splicing defects in prp43 mutants shows a very mild effect, similar to that of nonessential pre-mRNA splicing factors. Prp43p is the first DEXH/D-box protein shown to function in both RNA polymerase I and polymerase II transcript metabolism. Its essential function is in its newly characterized role in ribosome biogenesis of both ribosomal subunits, positioning Prp43p to regulate both pre-mRNA splicing and ribosome biogenesis. PMID:16382144

Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films.

PubMed Central

Pum, D; Weinhandl, M; Hödl, C; Sleytr, U B

1993-01-01

S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough. Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films. The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points. The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met. The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde. Images PMID:8478338

SAMe prevents the induction of the immunoproteasome and preserves the 26S proteasome in the DDC-induced MDB mouse model.

PubMed

Bardag-Gorce, Fawzia; Oliva, Joan; Li, Jun; French, Barbara A; French, Samuel W

2010-06-01

Mallory-Denk bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway's (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFalpha and IFNgamma receptors. SAMe prevented the increase in the TNFalpha and IFNgamma receptors, supporting the idea that TNFalpha and IFNgamma were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome. Copyright 2010 Elsevier Inc. All rights reserved.

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

PubMed

Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

2017-11-01

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABA A R could be a novel approach to treatment of hyperexcitability-related alterations in FXS. Copyright © 2017 Elsevier Inc. All rights reserved.

Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers

PubMed Central

Tsvetkov, Peter; Sokol, Ethan; Jin, Dexter; Brune, Zarina; Thiru, Prathapan; Ghandi, Mahmoud; Garraway, Levi A.; Gupta, Piyush B.; Santagata, Sandro; Whitesell, Luke; Lindquist, Susan

2017-01-01

The use of proteasome inhibitors to target cancer’s dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers. PMID:28028240

Small terminase couples viral DNA-binding to genome-packaging ATPase activity

PubMed Central

Roy, Ankoor; Bhardwaj, Anshul; Datta, Pinaki; Lander, Gabriel C.; Cingolani, Gino

2012-01-01

SUMMARY Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here, we describe the 1.75 Å crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ~23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of genome-packaging motors of general interest in virology. PMID:22771211

MALDI FTICR IMS of Intact Proteins: Using Mass Accuracy to Link Protein Images with Proteomics Data

NASA Astrophysics Data System (ADS)

Spraggins, Jeffrey M.; Rizzo, David G.; Moore, Jessica L.; Rose, Kristie L.; Hammer, Neal D.; Skaar, Eric P.; Caprioli, Richard M.

2015-06-01

MALDI imaging mass spectrometry is a highly sensitive and selective tool used to visualize biomolecules in tissue. However, identification of detected proteins remains a difficult task. Indirect identification strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. Here, we demonstrate the capabilities of MALDI FTICR MS for imaging intact proteins. MALDI FTICR IMS provides an unprecedented combination of mass resolving power (~75,000 at m/z 5000) and accuracy (<5ppm) for proteins up to ~12kDa, enabling identification based on correlation with LC-MS/MS proteomics data. Analysis of rat brain tissue was performed as a proof-of-concept highlighting the capabilities of this approach by imaging and identifying a number of proteins including N-terminally acetylated thymosin β4 ( m/z 4,963.502, 0.6ppm) and ATP synthase subunit ɛ ( m/z 5,636.074, -2.3ppm). MALDI FTICR IMS was also used to differentiate a series of oxidation products of S100A8 ( m/z 10,164.03, -2.1ppm), a subunit of the heterodimer calprotectin, in kidney tissue from mice infected with Staphylococcus aureus. S100A8 - M37O/C42O3 ( m/z 10228.00, -2.6ppm) was found to co-localize with bacterial microcolonies at the center of infectious foci. The ability of MALDI FTICR IMS to distinguish S100A8 modifications is critical to understanding calprotectin's roll in nutritional immunity.

Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Wostrikoff, Katia; Belcher, Susan; Barkan, Alice; Stern, David B

2012-08-01

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

PubMed

Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

1992-06-01

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Phosphorylation of Wheat Germ Initiation Factors and Ribosomal Proteins 1

PubMed Central

Browning, Karen S.; Yan, Tyan Fuh J.; Lauer, Stephen J.; Aquino, Lu Ann; Tao, Mariano; Ravel, Joanne M.

1985-01-01

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (Mr = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro. Images Fig. 1 Fig. 3 Fig. 4 PMID:16664060

Top-down proteomic identification of Shiga toxin 2 subtypes from Shiga toxin-producing Escherichia coli by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry.

PubMed

Fagerquist, Clifton K; Zaragoza, William J; Sultan, Omar; Woo, Nathan; Quiñones, Beatriz; Cooley, Michael B; Mandrell, Robert E

2014-05-01

We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)-tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.

Top-Down Proteomic Identification of Shiga Toxin 2 Subtypes from Shiga Toxin-Producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Tandem Time of Flight Mass Spectrometry

PubMed Central

Zaragoza, William J.; Sultan, Omar; Woo, Nathan; Quiñones, Beatriz; Cooley, Michael B.; Mandrell, Robert E.

2014-01-01

We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)–tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes. PMID:24584253

Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

PubMed Central

Chen, Chwen-Lih; Zhang, Liwen; Yeh, Alexander; Chen, Chun-An; Green-Church, Kari B.; Zweier, Jay L.; Chen, Yeong-Renn

2008-01-01

The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2·-) and O2·--derived oxidants change the redox status of the mitochondrial GSH pool. An electron transport protein, Mitochondrial Complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of Complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated Complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O2·- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact Complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of Complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibiton of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when Complex I is exposed to redox change in the GSH pool. PMID:17444656

Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae.

PubMed

Deguchi, T; Fukuoka, A; Yasuda, M; Nakano, M; Ozeki, S; Kanematsu, E; Nishino, Y; Ishihara, S; Ban, Y; Kawada, Y

1997-03-01

We determined a partial sequence of the Klebsiella pneumoniae parC gene, including the region analogous to the quinolone resistance-determining region of the Escherichia coli gyrA gene, and examined 26 clinical strains of K. pneumoniae for an association of alterations in GyrA and ParC with susceptibilities to quinolones. The study suggests that in K. pneumoniae DNA gyrase is a primary target of quinolones and that ParC alterations play a complementary role in the development of higher-level fluoroquinolone resistance.

Analyses of the Large Subunit Histidine-Rich Motif Expose an Alternative Proton Transfer Pathway in [NiFe] Hydrogenases

PubMed Central

Szőri-Dorogházi, Emma; Maróti, Gergely; Szőri, Milán; Nyilasi, Andrea; Rákhely, Gábor; Kovács, Kornél L.

2012-01-01

A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases. The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue. Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis. PMID:22511957

Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) β3 Subunits in Enhanced Functional Expression of Mouse α6β2β3- and α6β4β3-nAChRs*

PubMed Central

Dash, Bhagirathi; Li, Ming D.; Lukas, Ronald J.

2014-01-01

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where “*” indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5–8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′S subunits were substituted for mβ3V9′S subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops “C” (Glu221 and Phe223), “E” (Ser144 and Ser148), and “β2-β3” (Gln94 and Glu101) increased function of mα6mβ2*- (∼2–3-fold) or mα6mβ4* (∼2–4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs. PMID:25028511

Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

PubMed

Amino, Hisako; Osanai, Arihiro; Miyadera, Hiroko; Shinjyo, Noriko; Tomitsuka, Eriko; Taka, Hikari; Mineki, Reiko; Murayama, Kimie; Takamiya, Shinzaburo; Aoki, Takashi; Miyoshi, Hideto; Sakamoto, Kimitoshi; Kojima, Somei; Kita, Kiyoshi

2003-05-01

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

Fine genetic mapping of a gene for autosomal recessive retinitis pigmentosa on chromosome 6p21

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

Shugart, Yin Y.; Banerjee, P.; Knowles, J.A.

1995-08-01

The inherited retinal degenerations known as retinitis pigmentosa (RP) can be caused by mutations at many different loci and can be inherited as an autosomal recessive, autosomal dominant, or X-linked recessive trait. Two forms of autosomal recessive (arRP) have been reported to cosegregate with mutations in the rhodopsin gene and the beta-subunit of rod phosphodiesterase on chromosome 4p. Genetic linkage has been reported on chromosomes 6p and 1q. In a large Dominican family, we reported an arRp gene near the region of the peripherin/RDS gene. Four recombinations were detected between the disease locus and an intragenic marker derived from peripherin/RDS.more » 26 refs., 2 figs., 1 tab.« less

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency.

PubMed

Greene, Dina N; Whitney, Spencer M; Matsumura, Ichiro

2007-06-15

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.

Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

DOE R&D Accomplishments Database

Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

1982-06-01

This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

Molecular mechanics of 30S subunit head rotation.

PubMed

Mohan, Srividya; Donohue, John Paul; Noller, Harry F

2014-09-16

During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2.

Molecular mechanics of 30S subunit head rotation

PubMed Central

Mohan, Srividya; Donohue, John Paul; Noller, Harry F.

2014-01-01

During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2. PMID:25187561

Role of ART-27, a Novel Androgen Receptor Coactivator, in Normal Prostate and Prostate Cancer

DTIC Science & Technology

2005-04-01

associates with pro- teins that include RBP5, a subunit shared by RNA polymerases I, II , and Ill, an RBP5 binding protein called unconventional prefoldin ...of a large multiprotein complex that contains RNA polymerase II subunit 5, a subunit shared by all three RNA polymerases; unconventional prefoldin ...dithiothreitol; GRIP, glucocorticoid re- ceptor Interacting p rotein; HA, hemagglutinin; MMTV, mouse mamm ary tumor virus ; PAIS, partial AIS; SDS

[Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N

1997-05-01

The full-length cDNA of the rpc10+ gene encoding mini-subunit Rpc10, which is common for all three nuclear RNA polymerases of the fission yeast Schizosaccharomyces pombe, was cloned and sequenced. The Rpc10 subunit of Sz. pombe and its homologs from S. cerevisiae and H. sapiens are positively charged proteins with a highly conserved C-terminal region and an invariant zinc-binding domain (Zn-finger) of a typical amino acid composition: YxCx2Cx12RCx2CGxR. Functional tests of heterospecific complementation, using tetrad analysis or plasmid shuffling, showed that the Rpc10 subunit of Sz. pombe can successfully replace the homologous ABC10 alpha subunit in nuclear RNA polymerases I-III of S. cerevisiae.

Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay

PubMed Central

Li, Li; Yan, Hong-Bin; Blair, David; Lei, Meng-Tong; Cai, Jin-Zhong; Fan, Yan-Lei; Li, Jian-Qiu; Fu, Bao-Quan; Yang, Yu-Rong; McManus, Donald P.; Jia, Wan-Zhong

2015-01-01

Background Infections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool is needed to facilitate the detection of infected hosts and for species identification. Methodology/Principal Findings A single-tube multiplex PCR assay was established to differentiate the Echinococcus species responsible for infections in intermediate and definitive hosts. Primers specific for E. granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR accurately detected Echinococcus DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp. Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the multiplex PCR was 100% when evaluated using DNA isolated from other cestodes. Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E. multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with larval E. multilocularis and E. granulosus, respectively. Conclusions/Significance The multiplex PCR developed in this study is an efficient tool for discriminating E. granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis using feces collected from these definitive hosts. It can also be used for the identification of the Echinococcus metacestode larva in intermediate hosts, a stage that often cannot be identified to species on visual inspection. PMID:26393793

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

PubMed Central

Filbin, Megan E.; Kieft, Jeffrey S.

2011-01-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis. PMID:21606179

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

PubMed

Filbin, Megan E; Kieft, Jeffrey S

2011-07-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition

PubMed Central

Chia, Karin K. M.; Liu, Chia-Chi; Hamilton, Elisha J.; Garcia, Alvaro; Fry, Natasha A.; Hannam, William; Figtree, Gemma A.

2015-01-01

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na+-K+ pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na+-K+ pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na+-K+ pump current (Ip). Coexposure to 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na+-K+ pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47phox NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22phox subunit, and it decreased O2·−-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47phox indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22phox and p47phox NADPH oxidase subunits and decrease β1-Na+-K+ pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo. PMID:26084308

Molecular identification and description of the female of Nothoaspis reddelli (Ixodida: Argasidae) from a cave in southeastern Mexico.

PubMed

Guzmán-Cornejo, Carmen; Paredes-León, Ricardo; Labruna, Marcelo B; Nava, Santiago; Venzal, José M

2012-10-01

Nothoaspis reddelli Keirans and Clifford, 1975 , was described from 3 males collected in Grutas de Xtacumbilxunaán, Campeche, Mexico, although females have remained undescribed for 37 yr. Recently adult females of this species were collected from Cueva de Villa Luz ( =  Cueva de las Sardinas, Cueva del Azufre), in Tapijulapa, Tabasco, Mexico. Here we present a morphological description of the female stage, together with 16S rDNA sequences that confirm the conspecificity of our female, male, and nymphal specimens. The female integument of the anterior portion of the dorsal surface is smooth (nothoaspis), appearing to consist of 3 large "subunits," 1 anterior and 2 posterior, each with a small sublateral "subunit" on either side. The remaining dorsal covered integument is a cell-like configuration. The hood is large and bluntly rounded, and visible dorsally. The spiracular plate is oval. It possesses 1 pair of posthypostomal setae. The palpal trochanter has 1 pair of setae and a 5/5 hypostome decreasing to 4/4 at the apex. There is a single central pore at the base of the hypostome.

Genetic differentiation of the stingless bee Tetragonula pagdeni in Thailand using SSCP analysis of a large subunit of mitochondrial ribosomal DNA.

PubMed

Thummajitsakul, Sirikul; Klinbunga, Sirawut; Sittipraneed, Siriporn

2011-08-01

Genetic diversity and population differentiation of the stingless bee Tetragonula pagdeni (Schwarz) was assessed using single-strand conformational polymorphism (SSCP) analysis of a large subunit of the ribosomal RNA gene (16S rRNA). High levels of genetic variation among individuals within each population (North, Northeast, Central, Prachuap Khiri Khan, Chumphon, and Peninsular Thailand) of T. pagdeni were observed. Analysis of molecular variance indicated significant genetic differentiation among the six geographic populations (Φ (PT) = 0.28, P < 0.001) and between samples collected from north and south of the Isthmus of Kra (Φ (PT) = 0.18, P < 0.001). In addition, Φ (PT) values between all pairwise comparisons were statistically significant (P < 0.01), indicating strong degrees of intraspecific population differentiation. Therefore, PCR-SSCP is a simple and cost-effective technique applicable for routine population genetic analyses in T. pagdeni and other stingless bees. The results also provide an important baseline for the conservation and management of this ecologically important species.

Subunit mass analysis for monitoring antibody oxidation.

PubMed

Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

2017-04-01

Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

Immunohistochemical analyses of alpha1 and alpha3 Na+/K+-ATPase subunit expression in medulloblastomas.

PubMed

Suñol, Mariona; Cusi, Victoria; Cruz, Ofelia; Kiss, Robert; Lefranc, Florence

2011-03-01

The levels of expression of the α1 and α3 subunits of the Na(+)/K(+)-ATPase (the NaK sodium pump) in medulloblastomas are unclear. This study investigated the expression of the NaK subunits using immunohistochemical methods in 29 medulloblastomas including 23 classic, three large-cell/anaplastic and three nodular/desmoplastic medulloblastomas, as well as in three atypical teratoid/rhabdoid tumors (AT/RTs). There was overexpression of the α1 or α3 NaK subunits in more than half of the medulloblastomas and atypical AT/RTs, with about one-third of these tumours displaying overexpression of both subunits. These preliminary data suggest that targeting these subunits in AT/RTs and medulloblastomas that overexpress these proteins may lead to therapeutic benefit. These findings warrant confirmation in larger numbers of patients than those used in this study. Moreover, it should be determined whether inhibition of the α1/α3 NaK subunits can be integrated into the risk stratification schemes already in use for medulloblastoma patients.

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

PubMed Central

2014-01-01

Background The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. Findings This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. Conclusions In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle. PMID:25379053

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

PubMed

Uzunova, Sonya Dimitrova; Zarkov, Alexander Stefanov; Ivanova, Anna Marianova; Stoynov, Stoyno Stefanov; Nedelcheva-Veleva, Marina Nedelcheva

2014-01-01

The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

DiBAC4(3) hits a “sweet spot” for the activation of arterial large-conductance Ca2+-activated potassium channels independently of the β1-subunit

PubMed Central

Scornik, Fabiana S.; Bucciero, Ronald S.; Wu, Yuesheng; Selga, Elisabet; Bosch Calero, Cristina; Brugada, Ramon

2013-01-01

The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] has been reported as a novel large-conductance Ca2+-activated K+ (BK) channel activator with selectivity for its β1- or β4-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory β1-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the β1-subunit, we explored the effects of DiBAC4(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC4(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic β1-knockout mice in excised patches. BK channels from brain arteries, with or without the β1-subunit, were similarly activated by DiBAC4(3). In addition, we found that saturating concentrations of DiBAC4(3) (∼30 μM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as −300 mV. This “sweet spot” for persistent activation is independent of Ca2+ and/or the β1–4-subunits and is fully achieved when DiBAC4(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC4(3) varies across species and/or vascular beds. DiBAC4(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators. PMID:23542916

Inter-subunit electrostatic interactions in ferritin molecule: comparison with inter-molecular interactions in crystals

NASA Astrophysics Data System (ADS)

Takahashi, Takuya; Hogyoku, Michiru; Nagayama, Kuniaki

1996-10-01

We evaluated the contribution of electrostatic interactions to the stability of macromolecular assembly in a horse L ferritin molecule composed of 24 subunits and the three-dimensional crystal of the ferritin molecules with numerical calculation of Poisson-Boltzmann equation based on dielectric model. The calculation showed that the electrostatic energy both favors the assembly of the 24 subunits and the crystalline assembly of the ferritin molecules (i.e., 24-mers). Short-range interactions less than 5 Å such as salt bridges and hydrogen bonds were important for both the subunit assembly and the crystalline assembly. To elucidate the strong stabilization by electrostatic interactions in both the ferritin 24-mer and its crystal, we analyzed the contribution of individual atoms. It revealed that the stabilization was arising from buried salt bridges or hydrogen bonds, which yielded more than 5 kcal/mol in some interactions. These large electrostatic stabilization and also the unexpected small ionic strength dependence was different from those of bovine pancreatic trypsin inhibitor (BPTI) orthorhombic and pig-insulin cubic crystals previously calculated. We also evaluated changes of the accessible surface area (ASA) and hydration free energy in accordance with the process of the subunit assembly. The change of hydration free energy, which was very large (i.e. ˜ + 100 kcal/mol/subunit) and unfavorable for the assembly, was proportional to the electrostatic hydration energy (i.e. Born energy change in hydration process). Hydrophobic groups were likely to appear more frequently than hydrophilic groups at the subunit interfaces. These results suggest that the molecular structure of the ferritin 24-mer and the crystal structure of the 24-mers were both stabilized by local electrostatic interactions, in particular. We view protein crystals as an extension of the protein oligomer to an infinite number of subunits association.

Subunit mass fingerprinting of mitochondrial complex I.

PubMed

Morgner, Nina; Zickermann, Volker; Kerscher, Stefan; Wittig, Ilka; Abdrakhmanova, Albina; Barth, Hans-Dieter; Brutschy, Bernhard; Brandt, Ulrich

2008-10-01

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism

DOE PAGES

VanderLinden, Ryan T.; Hemmis, Casey W.; Yao, Tingting; ...

2017-04-25

This work presents that the 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to themore » C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. In conclusion, these findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics.« less

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism.

PubMed

VanderLinden, Ryan T; Hemmis, Casey W; Yao, Tingting; Robinson, Howard; Hill, Christopher P

2017-06-09

The 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to the C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. These findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal structure studies of NADP{sup +} dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

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

Kumar, S.M.; Pampa, K.J.; Manjula, M.

2014-06-20

Highlights: • We determined the structure of isocitrate dehydrogenase with citrate and cofactor. • The structure reveals a unique novel terminal domain involved in dimerization. • Clasp domain shows significant difference, and catalytic residues are conserved. • Oligomerization of the enzyme is quantized with subunit-subunit interactions. • Novel domain of this enzyme is classified as subfamily of the type IV. - Abstract: NADP{sup +} dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP{supmore » +} was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.« less

Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study.

PubMed

Hauer, J A; Taylor, S S; Johnson, D A

1999-05-25

The conformational flexibility of peptidyl ligands may be an essential element of many peptide-macromolecular interactions. Consequently, the alpha-carbonyl backbone flexibility of the 8 kDa protein kinase inhibitor (PKI alpha) peptide of cAMP-dependent protein kinase (cAPK) free in solution and bound to cAPK was assessed by time-resolved fluorescence anisotropy. Specifically, three full-length, single-site PKI alpha mutants (V3C, S28C, and S59C) were prepared, and fluorescein iodoacetamide (FI) was selectively conjugated to the side chains of each substituted cysteine. The time-resolved anisotropy decay profiles of the labeled mutants were well fit to a model-free nonassociative biexponential equation. Free in solution, the three labeled proteins had very similar anisotropy decays arising primarily from local alpha-carbonyl backbone movements. Only a small fraction of the anisotropy decay was associated with slower, whole-body tumbling, confirming that PKI alpha is highly disordered at all three locations. Complexation of the mutants with the catalytic (C) subunit of cAPK decreased the rate of whole-body tumbling for all three mutants. The effects on the rapid decay processes, however, were dependent upon the site of conjugation. The anisotropy decay profiles of both FI-V3C- and FI-S28C-PKI alpha were associated with significantly reduced contributions from the fast decay processes, while that of FI-S59C-PKI alpha was largely unaffected by binding to the C-subunit. The results suggest that the cAPK-binding domain of PKI alpha extends from the its N-terminus to residues beyond Ser28 but does not include the segment around Ser59, which is still part of a highly flexible domain when bound to the C-subunit.

Increasing the starch content and grain weight of common wheat by overexpression of the cytosolic AGPase large subunit gene.

PubMed

Kang, Guozhang; Liu, Guoqin; Peng, Xiaoqi; Wei, Liting; Wang, Chenyang; Zhu, YunJi; Ma, Ying; Jiang, Yumei; Guo, Tiancai

2013-12-01

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of starch synthesis. AGPase is a heterotetramer composed of two large subunits and two small subunits, has cytosolic and plastidial isoforms, and is detected mainly in the cytosol of endosperm in cereal crops. To investigate the effects of AGPase cytosolic large subunit gene (LSU I) on starch biosynthesis in higher plant, in this study, a TaLSU I gene from wheat was overexpressed under the control of an endosperm-specific promoter in a wheat cultivar (Yumai 34). PCR, Southern blot, and real-time RT-PCR analyses indicated that the transgene was integrated into the genome of transgenic plants and was overexpressed in their progeny. The overexpression of the TaLSU I gene remarkably enhanced AGPase activity, endosperm starch weight, grain number per spike, and single grain weight. Therefore, we conclude that overexpression of the TaLSU I gene enhances the starch biosynthesis in endosperm of wheat grains, having potential applications in wheat breeding to develop a high-yield wheat cultivar with high starch weight and kernel weight. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Two hydrophobic subunits are essential for the heme b ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli.

PubMed

Nakamura, K; Yamaki, M; Sarada, M; Nakayama, S; Vibat, C R; Gennis, R B; Nakayashiki, T; Inokuchi, H; Kojima, S; Kita, K

1996-01-05

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b556. An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b556 in its CO reactivity and red shift of the alpha absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinate-ubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquitnone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.

The gamma subunit of transducin is farnesylated.

PubMed Central

Lai, R K; Perez-Sala, D; Cañada, F J; Rando, R R

1990-01-01

Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification. Images PMID:2217200

Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3.

PubMed Central

Ehresmann, C; Moine, H; Mougel, M; Dondon, J; Grunberg-Manago, M; Ebel, J P; Ehresmann, B

1986-01-01

The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far. Images PMID:2425339

Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting.

PubMed Central

Clarkson, G H; Neagle, J; Lindsay, J G

1991-01-01

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1996968

The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule.

PubMed Central

Barrett, A J; Brown, M A; Sayers, C A

1979-01-01

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact alpha 2M. F-alpha 2M was less easily dissociated than was S-alpha 2M. S-alpha 2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Inact reactive alpha 2M could then be regenerated in high yield by reoxidation of the subunits. F-alpha 2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein... Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:91367

Conformational Sampling and Nucleotide-Dependent Transitions of the GroEL Subunit Probed by Unbiased Molecular Dynamics Simulations

PubMed Central

Skjaerven, Lars; Grant, Barry; Muga, Arturo; Teigen, Knut; McCammon, J. Andrew; Reuter, Nathalie; Martinez, Aurora

2011-01-01

GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant “opening” of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition. PMID:21423709

Purification and properties of a third form of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the enterobacteriaceae

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

Largen, M.; Mills, S.E.; Rowe, J.

1978-01-25

Anthranilate-5-phosphoribosypyrophosphate phosphoribosyltransferase was purified from the bacterium Erwinia carotovora, a member of the Enterobacteriaceae. The enzyme was homogeneous according to the criteria of gel electrophoresis and NH/sub 2/-terminal amino acid sequence analysis. The molecular weight of the enzyme as determined on a calibrated Sephadex G-200 column was 67,000 +- 2,000. Sodium dodecyl sulfate-polyacrylamide gels gave a subunit molecular weight of 40,000 +- 1,000, suggesting that the enzyme was a dimer. A comparison of the NH/sub 2/-terminal sequence of the enzyme with the (previously determined) homologue from Serratia marcescens, a monomer with a molecular weight of 45,000, showed that the largermore » Serratia subunit came into register with amino acid 14 of the Erwinia subunit. The register for the length of the known overlap, 26 amino acids, was highly conserved.« less

Identification of functional domains within the alpha and beta subunits of beta-hexosaminidase A through the expression of alpha-beta fusion proteins.

PubMed

Tse, R; Wu, Y J; Vavougios, G; Hou, Y; Hinek, A; Mahuran, D J

1996-08-20

There are three human beta-hexosaminidase isozymes which are composed of all possible dimeric combinations of an alpha and/or a beta subunit; A (alpha beta), and B (beta beta), and S (alpha alpha). The amino acid sequences of the two subunits are 60% identical. The homology between the two chains varies with the middle > the carboxy-terminal > > the amino-terminal portions. Although dimerization is required for activity, each subunit contains its own active site and differs in its substrate specificity and thermal stability. The presence of the beta subunit in hexosaminidase A also influences the substrate specificity of the alpha subunit; e.g., in vivo only the A heterodimer can hydrolyze GM2 ganglioside. In this report, we localize functional regions in the two subunits by cellular expression of alpha/beta fusion proteins joined at adjacently aligned residues. First, a chimeric alpha/beta chain was made by replacing the least well-conserved amino-terminal section of the beta chain with the corresponding alpha section. The biochemical characteristics of this protein were nearly identical to hexosaminidase B. Therefore, the most dissimilar regions in the subunits are not responsible for their dissimilar biochemical properties. A second fusion protein was made that also included the more homologous middle section of the alpha chain. This protein expressed the substrate specificity unique to isozymes containing an alpha subunit (A and S). We conclude that the region responsible for the ability of the alpha subunit to bind negatively charged substrates is located within residues alpha 132-283. Interestingly, the remaining carboxy-terminal section from the beta chain, beta 316-556, was sufficient to allow this chimera to hydrolyze GM2 ganglioside with 10% the specific activity of heterodimeric hexosaminidase A. Thus, the carboxy-terminal section of each subunit is likely involved in subunit-subunit interactions.

Cryphodera sinensis n. sp. (Nematoda: Heteroderidae), a non-cyst-forming parasitic nematode from the root of ramie Boehmeria nivea in China.

PubMed

Zhuo, K; Wang, H H; Ye, W; Peng, D L; Liao, J L

2014-12-01

Cryphodera sinensis n. sp. is described from ramie (Boehmeria nivea) based on the morphology and molecular analyses of rRNA small subunit (SSU), D2D3 expansion domains of large subunit (LSU D2D3) and internal transcribed spacer (ITS). This new species is characterized by oval females with a distinct subcrystalline layer and pronounced and protruding vulval lip, distinctly concave vulva-anus profile and a vulva-anus distance of 29.5-35.8 μm. Males possess two annuli in the lip region, a stylet 27-32.5 μm in length with round knobs sloping slightly posteriorly, lateral fields with three lines, spicules 20-28 μm long and the presence of a short cloacal tube. Second-stage juveniles possess three lip annuli, a stylet 28-31 μm in length with well-developed knobs projected anteriorly and three lines along the lateral field. The pointed tail, 52-65 μm long, possesses a mucro-like tip and a hyaline region, 24.5-35 μm long. Large phasmids with a lens-like structure are located 2-6 annuli posterior to the anus. Phylogenetic analysis shows that the species has unique SSU, LSU D2D3 and ITS rRNA sequences. Phylogenetic relationships of the three rDNA sequences of C. sinensis n. sp. and other cystoid/cyst nematodes are analysed together with a comparison of other species within the genus Cryphodera.

Purification and Characterization of Hemagglutinating Proteins from Poker-Chip Venus (Meretrix lusoria) and Corbicula Clam (Corbicula fluminea)

PubMed Central

Cheng, Chin-Fu; Hung, Shao-Wen; Chang, Yung-Chung; Chen, Ming-Hui; Chang, Chen-Hsuan; Tsou, Li-Tse; Tu, Ching-Yu; Lin, Yu-Hsing; Liu, Pan-Chen; Lin, Shiun-Long; Wang, Way-Shyan

2012-01-01

Hemagglutinating proteins (HAPs) were purified from Poker-chip Venus (Meretrix lusoria) and Corbicula clam (Corbicula fluminea) using gel-filtration chromatography on a Sephacryl S-300 column. The molecular weights of the HAPs obtained from Poker-chip Venus and Corbicula clam were 358 kDa and 380 kDa, respectively. Purified HAP from Poker-chip Venus yielded two subunits with molecular weights of 26 kDa and 29 kDa. However, only one HAP subunit was purified from Corbicula clam, and its molecular weight was 32 kDa. The two Poker-chip Venus HAPs possessed hemagglutinating ability (HAA) for erythrocytes of some vertebrate animal species, especially tilapia. Moreover, HAA of the HAP purified from Poker-chip Venus was higher than that of the HAP of Corbicula clam. Furthermore, Poker-chip Venus HAPs possessed better HAA at a pH higher than 7.0. When the temperature was at 4°C–10°C or the salinity was less than 0.5‰, the two Poker-chip Venus HAPs possessed better HAA compared with that of Corbicula clam. PMID:22666167

Recognition by nonaromatic and stereochemical subunit-containing polyamides of the four Watson-Crick base pairs in the DNA minor groove.

PubMed

Zhang, Hong-Fei; Wu, Yan-Ling; Jiang, Shi-Kun; Wang, Pu; Sugiyama, Hiroshi; Chen, Xing-Lai; Zhang, Wen; Ji, Yan-Juan; Guo, Chuan-Xin

2012-06-18

In order to develop an optimal subunit as a T-recognition element in hairpin polyamides, 15 novel chirality-modified polyamides containing (R)-α,β-diaminopropionic acid ((R) β α-NH 2), (S)-α,β-diaminopropionic acid ((S) β α-NH 2), (1R,3S)-3-aminocyclopentanecarboxylic acid ((RS) Cp), (1S,3R)-3-amino-cyclopentanecarboxylic acid ((RS) Cp), (1R,3R)-3-aminocyclopentanecarboxylic acid ((RR) Cp) and (1S,3S)-3-amino-cyclopentanecarboxylic acid ((SS) Cp) residues were synthesized. Their binding characteristics to DNA sequences 5'-TGCNCAT-3'/3'-ACGN'GTA-5' (N⋅N'=A⋅T, T⋅A, G⋅C and C⋅G) were systemically studied by surface plasmon resonance (SPR) and molecular simulation (MSim) techniques. SPR showed that polyamide 4, AcIm-(S) β α-NH 2-ImPy-γ-ImPy-β-Py-βDp (β/(S) β α-NH 2 pair), bound to a DNA sequence containing a core binding site of 5'-TGCACAT-3' with a dissociation equilibrium constant (K(D) ) of 4.5×10(-8)  m. This was a tenfold improvement in specificity over 5'-TGCTCAT-3' (K(D) =4.5×10(-7)  M). MSim studies supported the SPR results. More importantly, for the first time, we found that chiral 3-aminocyclopentanecarboxylic acids in polyamides can be employed as base readers with only a small decrease in binding affinity to DNA. In particular, SPR showed that polyamide 9 ((RR) Cp/β pair) had a 15-fold binding preference for 5'-TGCTCAT-3' over 5'-TGCACAT-3'. A large difference in standard free energy change for A⋅T over T⋅A was determined (ΔΔG(o) =5.9 kJ mol(-1) ), as was a twofold decrease in interaction energy by MSim. Moreover, a 1:1 stoichiometry (9 to 5'-TGCTCAT-3'/3'-ACGAGTA-5') was shown by MSim to be optimal for the chiral five-membered cycle to fit the minor groove. Collectively, the study suggests that the (S)-α-amino-β-aminopropionic acid and (1R,3R)-3-aminocyclopentanecarboxylic acid can serve as a T-recognition element, and the stereochemistry and the nature of these subunits significantly influence binding properties in these recognition events. Subunit (1R,3R)-3-aminocyclopentanecarboxylic acid broadens our scope to design novel polyamides. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants

PubMed Central

Abbott, Geoffrey W.

2017-01-01

The human ventricular cardiomyocyte transient outward K+ current (Ito) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac Ito is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology. PMID:28228734

β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants.

PubMed

Abbott, Geoffrey W

2017-01-01

The human ventricular cardiomyocyte transient outward K + current ( I to ) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac I to is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology.

A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Belcher, Susan; Montano, Monica; Barkan, Alice; Stern, David B

2014-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

β-hydroxy-β-methylbutyrate (HMB) prevents sepsis-induced diaphragm dysfunction in mice.

PubMed

Supinski, Gerald S; Callahan, Leigh A

2014-06-01

Infections induce severe respiratory muscle weakness. Currently there are no treatments for this important clinical problem. We tested the hypothesis that β-hydroxy-β-methylbutyrate (HMB) would prevent sepsis-induced diaphragm weakness. Four groups of adult male mice were studied: controls (saline-injected), sepsis (intraperitoneal lipopolysaccharide), sepsis+HMB (injected intravenously), and HMB. Diaphragm force generation and indices of caspase 3, calpain, 20S proteasomal subunit, and double-stranded RNA-dependent protein kinase (PKR) activation were assessed after 24h. Sepsis elicited large reductions in diaphragm specific force generation at all stimulation frequencies. Endotoxin also activated caspase 3, calpain, the 20S proteasomal subunit and PKR in the diaphragm. HMB blocked sepsis-induced caspase 3, 20S proteasomal and PKR activation, but did not prevent calpain activation. Most importantly, HMB administration significantly attenuated sepsis-induced diaphragm weakness, preserving muscle force generation at all stimulation frequencies (p<0.01). We speculate that HMB may prove to be an important therapy in infected patients, with the potential to increase diaphragm strength, to reduce the duration of mechanical ventilation and to decrease mortality in this patient population. Copyright © 2014 Elsevier B.V. All rights reserved.

D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp.

PubMed

Dagar, Sumit S; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K

2011-09-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize[C][W

PubMed Central

Feiz, Leila; Williams-Carrier, Rosalind; Wostrikoff, Katia; Belcher, Susan; Barkan, Alice; Stern, David B.

2012-01-01

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process. PMID:22942379

Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp. Strain T7-7

PubMed Central

Li, Ping; Wang, Lei

2013-01-01

The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium. PMID:23417490

Structural changes of homodimers in the PDB.

PubMed

Koike, Ryotaro; Amemiya, Takayuki; Horii, Tatsuya; Ota, Motonori

2018-04-01

Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of potential diatom 'barcode' genes (the 18S rRNA gene and ITS, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in the Bacillariophyta.

PubMed

Guo, Liliang; Sui, Zhenghong; Zhang, Shu; Ren, Yuanyuan; Liu, Yuan

2015-04-01

Diatoms form an enormous group of photoautotrophic micro-eukaryotes and play a crucial role in marine ecology. In this study, we evaluated typical genes to determine whether they were effective at different levels of diatom clustering analysis to assess the potential of these regions for barcoding taxa. Our test genes included nuclear rRNA genes (the nuclear small-subunit rRNA gene and the 5.8S rRNA gene+ITS-2), a mitochondrial gene (cytochrome c-oxidase subunit 1, COI), a chloroplast gene [ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL)] and the universal plastid amplicon (UPA). Calculated genetic divergence was highest for the internal transcribed spacer (ITS; 5.8S+ITS-2) (p-distance of 1.569, 85.84% parsimony-informative sites) and COI (6.084, 82.14%), followed by the 18S rRNA gene (0.139, 57.69%), rbcL (0.120, 42.01%) and UPA (0.050, 14.97%), which indicated that ITS and COI were highly divergent compared with the other tested genes, and that their nucleotide compositions were variable within the whole group of diatoms. Bayesian inference (BI) analysis showed that the phylogenetic trees generated from each gene clustered diatoms at different phylogenetic levels. The 18S rRNA gene was better than the other genes in clustering higher diatom taxa, and both the 18S rRNA gene and rbcL performed well in clustering some lower taxa. The COI region was able to barcode species of some genera within the Bacillariophyceae. ITS was a potential marker for DNA based-taxonomy and DNA barcoding of Thalassiosirales, while species of Cyclotella, Skeletonema and Stephanodiscus gathered in separate clades, and were paraphyletic with those of Thalassiosira. Finally, UPA was too conserved to serve as a diatom barcode. © 2015 IUMS.

Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.

PubMed

Tsvetkov, Peter; Sokol, Ethan; Jin, Dexter; Brune, Zarina; Thiru, Prathapan; Ghandi, Mahmoud; Garraway, Levi A; Gupta, Piyush B; Santagata, Sandro; Whitesell, Luke; Lindquist, Susan

2017-01-10

The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.

Identification of epitopes of the β subunit of soybean β-conglycinin that are antigenic in pigs, dogs, rabbits and fish.

PubMed

Taliercio, Earl; Loveless, Telisa M; Turano, Marc J; Kim, Sung Woo

2014-08-01

β-Conglycinin (conglycinin) is one of the major seed storage proteins of soybean. Conglycinin is a 7S trimer composed of different combinations of β, α and α' subunits. All subunits of conglycinin have been reported to be allergenic in humans. The goal of this research is to identify epitopes of the β subunit of conglycinin that are antigenic in multiple animal species. Sera from pigs, dogs, rabbits and hybrid striped bass that had antibodies against soybean conglycinin were identified by ELISA. Most of these sera recognized peptides that represent the β subunit of conglycinin. One antigenic region of the β subunit of conglycinin had considerable overlap among all species tested. One region that was similar to a peanut allergenic epitope in humans overlapped with a region that binds IgE from dogs. One region was antigenic in multiple rabbits and pigs, suggesting it may play a role in the response of pigs to soybean in the diet. One region of the β subunit of conglycinin is an important antigen across species and abuts a region similar to the peanut allergen ARA h 1. A second region is particularly antigenic in pigs and rabbits. Variants of these antigenic regions of the β subunit of conglycinin may be useful in determining the role these regions play in the health of animals fed soybean. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Crystal structures of the adenylate sensor from fission yeast AMP-activated protein kinase.

PubMed

Townley, Robert; Shapiro, Lawrence

2007-03-23

The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular ATP (adenosine triphosphate) and AMP concentrations. Here, we report crystal structures at 2.9 and 2.6 A resolution for ATP- and AMP-bound forms of a core alphabetagamma adenylate-binding domain from the fission yeast AMPK homolog. ATP and AMP bind competitively to a single site in the gamma subunit, with their respective phosphate groups positioned near function-impairing mutants. Unexpectedly, ATP binds without counterions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

PubMed Central

Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

2013-01-01

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

PubMed Central

Courtot, Elise; Charvet, Claude L.; Beech, Robin N.; Harmache, Abdallah; Wolstenholme, Adrian J.; Holden-Dye, Lindy; O’Connor, Vincent; Peineau, Nicolas; Woods, Debra J.; Neveu, Cedric

2015-01-01

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

Diverse Roles for Auxiliary Subunits in Phosphorylation-Dependent Regulation of Mammalian Brain Voltage-Gated Potassium Channels

PubMed Central

Vacher, Helene; Trimmer, James S.

2012-01-01

Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse, in part due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons, and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself, or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels. PMID:21822597

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae)

PubMed Central

Straub, Shannon C.K.; Fishbein, Mark; Liston, Aaron

2015-01-01

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming). PMID:25653903

Expression of NMDA receptor subunits in human blood lymphocytes: A peripheral biomarker in online computer game addiction.

PubMed

Sadat-Shirazi, Mitra-Sadat; Vousooghi, Nasim; Alizadeh, Bentolhoda; Makki, Seyed Mohammad; Zarei, Seyed Zeinolabedin; Nazari, Shahrzad; Zarrindast, Mohammad Reza

2018-05-23

Background and aims Repeated performance of some behaviors such as playing computer games could result in addiction. The NMDA receptor is critically involved in the development of behavioral and drug addictions. It has been claimed that the expression level of neurotransmitter receptors in the brain may be reflected in peripheral blood lymphocytes (PBLs). Methods Here, using a real-time PCR method, we have investigated the mRNA expression of GluN2A, GluN2D, GluN3A, and GluN3B subunits of the NMDA receptor in PBLs of male online computer game addicts (n = 25) in comparison with normal subjects (n = 26). Results Expression levels of GluN2A, GluN2D, and GluN3B subunits were not statistically different between game addicts and the control group. However, the mRNA expression of the GluN3A subunit was downregulated in PBLs of game addicts. Discussion and conclusions Transcriptional levels of GluN2A and GluN2D subunits in online computer game addicts are similar to our previously reported data of opioid addiction and are not different from the control group. However, unlike our earlier finding of drug addiction, the mRNA expression levels of GluN3A and GluN3B subunits in PBLs of game addicts are reduced and unchanged, respectively, compared with control subjects. It seems that the downregulated state of the GluN3A subunit of NMDA receptor in online computer game addicts is a finding that deserves more studies in the future to see whether it can serve as a peripheral biomarker in addiction studies, where the researcher wants to rule out the confusing effects of abused drugs.

Many functions of the meiotic cohesin.

PubMed

Bardhan, Amit

2010-12-01

Sister chromatids are held together from the time of their formation in S phase until they segregate in anaphase by the cohesin complex. In meiosis of most organisms, the mitotic Mcd1/Scc1/Rad21 subunit of the cohesin complex is largely replaced by its paralog named Rec8. This article reviews the specialized functions of Rec8 that are crucial for diverse aspects of chromosome dynamics in meiosis, and presents some speculations relating to meiotic chromosome organization.

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

PubMed

Toh, Seok-Ming; Xiong, Liqun; Arias, Cesar A; Villegas, Maria V; Lolans, Karen; Quinn, John; Mankin, Alexander S

2007-06-01

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

PubMed Central

Arenz, Stefan; Graf, Michael; Nguyen, Fabian; Huter, Paul; Polikanov, Yury S.; Blanchard, Scott C.; Wilson, Daniel N.

2016-01-01

The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis. PMID:27330110

Structure and variation of the mitochondrial genome of fishes.

PubMed

Satoh, Takashi P; Miya, Masaki; Mabuchi, Kohji; Nishida, Mutsumi

2016-09-07

The mitochondrial (mt) genome has been used as an effective tool for phylogenetic and population genetic analyses in vertebrates. However, the structure and variability of the vertebrate mt genome are not well understood. A potential strategy for improving our understanding is to conduct a comprehensive comparative study of large mt genome data. The aim of this study was to characterize the structure and variability of the fish mt genome through comparative analysis of large datasets. An analysis of the secondary structure of proteins for 250 fish species (248 ray-finned and 2 cartilaginous fishes) illustrated that cytochrome c oxidase subunits (COI, COII, and COIII) and a cytochrome bc1 complex subunit (Cyt b) had substantial amino acid conservation. Among the four proteins, COI was the most conserved, as more than half of all amino acid sites were invariable among the 250 species. Our models identified 43 and 58 stems within 12S rRNA and 16S rRNA, respectively, with larger numbers than proposed previously for vertebrates. The models also identified 149 and 319 invariable sites in 12S rRNA and 16S rRNA, respectively, in all fishes. In particular, the present result verified that a region corresponding to the peptidyl transferase center in prokaryotic 23S rRNA, which is homologous to mt 16S rRNA, is also conserved in fish mt 16S rRNA. Concerning the gene order, we found 35 variations (in 32 families) that deviated from the common gene order in vertebrates. These gene rearrangements were mostly observed in the area spanning the ND5 gene to the control region as well as two tRNA gene cluster regions (IQM and WANCY regions). Although many of such gene rearrangements were unique to a specific taxon, some were shared polyphyletically between distantly related species. Through a large-scale comparative analysis of 250 fish species mt genomes, we elucidated various structural aspects of the fish mt genome and the encoded genes. The present results will be important for understanding functions of the mt genome and developing programs for nucleotide sequence analysis. This study demonstrated the significance of extensive comparisons for understanding the structure of the mt genome.

Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit.

PubMed Central

Yang, F; Curran, S C; Li, L S; Avarbock, D; Graf, J D; Chua, M M; Lu, G; Salem, J; Rubin, H

1997-01-01

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon. PMID:9335290

Differential regulation of luteinizing hormone and follicle-stimulating hormone expression during ovarian development and under sexual steroid feedback in the European eel.

PubMed

Schmitz, Monika; Aroua, Salima; Vidal, Bernadette; Le Belle, Nadine; Elie, Pierre; Dufour, Sylvie

2005-01-01

Pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are, in teleosts as in mammals, under the control of hypothalamic factors and steroid feedbacks. In teleosts, feedback regulations largely vary depending on species and physiological stage. In the present study the regulation of FSH and LH expression was investigated in the European eel, a fish of biological and phylogenetical interest as a representative of an early group of teleosts. The eel FSHbeta subunit was cloned, sequenced and together with earlier isolated eel LHbeta and glycoprotein hormone alpha (GPalpha) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHbeta and LHbeta are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHbeta and FSHbeta subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHbeta and GPalpha increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHbeta mRNA levels decreased. To understand the role of sex steroids in these opposite variations, immature eels were treated with estradiol (E2)and testosterone (T), both steroids being produced in eel ovaries during gonadal development. E2 treatment induced increases in both LHbeta and GPalpha mRNA levels, without any significant effect on FSHbeta. In contrast, T treatment induced a decrease in FSHbeta mRNA levels, without any significant effect on the other subunits. These data demonstrate that steroids exert a differential feedback on eel gonadotropin expression, with an E2-specific positive feedback on LH and a T-specific negative feedback on FSH, leading to an opposite regulation of LH and FSH during ovarian development. Copyright 2005 S. Karger AG, Basel

Chaperones of F[subscript 1]-ATPase

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

Ludlam, Anthony; Brunzelle, Joseph; Pribyl, Thomas

2009-09-25

Mitochondrial F{sub 1}-ATPase contains a hexamer of alternating {alpha} and {beta} subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to {beta} and {alpha}. In the absence of Atp11p and Atp12p, the hexamer is not formed, and {alpha} and {beta} precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F{sub 1} assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486--1493) hypothesized that the chaperones themselves look very much like the {alpha} and {beta} subunits, and proposed an exchange of Atp11pmore » for {alpha} and of Atp12p for {beta}; the driving force for the exchange was expected to be a higher affinity of {alpha} and {beta} for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to {beta} and Atp12p is bound to {alpha}, the two F{sub 1} subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to {alpha} and {beta} prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble {alpha} or {beta}, and it is instead the F{sub 1} {gamma} subunit that initiates the release of the chaperones from {alpha} and {beta} and their further assembly into the mature complex.« less

Molecular phylogeny of Laetiporus and other brown rot polypore genera in North America

Treesearch

Daniel L. Lindner; Mark T. Banik

2008-01-01

Phylogenetic relationships were investigated among North American species of Laetiporus, Leptoporus, Phaeolus, Pycnoporellus, and Wolfiporia using ITS, nuclear large subunit and mitochondrial small subunit rDNA sequences. Members of these genera have poroid hymenophores, simple septate hyphae and cause brown rots in a variety of...

D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp.▿

PubMed Central

Dagar, Sumit S.; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K.

2011-01-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation. PMID:21784906

Sequence data for two large-subunit rRNA genes from an Asian strain of Alexandrium catenella.

PubMed Central

Yeung, P K; Kong, K F; Wong, F T; Wong, J T

1996-01-01

PCR generated two distinct products from a toxic isolate of Alexandrium catenella, which had been taken from Dai Ya Bay (southern China), by using primers for large-subunit rRNA. This pattern is distinct from published data for North American Alexandrium species. Sequences of the two products suggest that the smaller was generated by a deletion event. Single-cell PCR generated the same pattern, confirming that the two products were not the results from different individuals. PMID:8900010

A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome.

PubMed

Netterling, Sakura; Vaitkevicius, Karolis; Nord, Stefan; Johansson, Jörgen

2012-08-01

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.

LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes.

PubMed

Mund, Markus; Overbeck, Jan H; Ullmann, Janina; Sprangers, Remco

2013-10-18

Seeing the big picture: Asymmetric macromolecular complexes that are NMR active in only a subset of their subunits can be prepared, thus decreasing NMR spectral complexity. For the hetero heptameric LSm1-7 and LSm2-8 rings NMR spectra of the individual subunits of the complete complex are obtained, showing a conserved RNA binding site. This LEGO-NMR technique makes large asymmetric complexes accessible to detailed NMR spectroscopic studies. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of Creative Commons the Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Stopped-flow studies of changes in fluorescence of 8-anilino-1-naphthalene sulfonic acid caused by magnesium and salt binding to yeast enolase.

PubMed

Brewer, J M

1976-12-11

Stopped-flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8-anilino-1-naphthalenesulfonic acid fluorescence changes. The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped-flow measurements. These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates. From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation.

Plastid transformation for Rubisco engineering and protocols for assessing expression.

PubMed

Whitney, Spencer M; Sharwood, Robert E

2014-01-01

The assimilation of CO2 within chloroplasts is catalyzed by the bi-functional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multi-gene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a long-term goal which has been greatly advanced in recent times using plastid transformation techniques. Here we present experimental methodologies for efficiently engineering Rubisco in the plastids of a tobacco master-line and analyzing leaf Rubisco content.

[Molecular farming has come of age].

PubMed

Chiao, J S

2001-07-01

Important development in plant biotechnology has been ushered in the 1990s, the most astounding field is the establishment of various transgenic crops, including cotton, corn, rice, tomato etc. In addition, utilization of transgenic plants for the generation of antibodies, gene epitopes and complex proteins has drawn much attention recently. Among these results, the use of plant parts with inducted antigens such as cholera toxin B subunit, toxigenic E. coli LT-B subunit as oral vaccines is attractive as a new route of medication. Considering their effectiveness as vaccines in animal and clinical tests, and simple agricultural practice for large scale production it is anticipated that these antibodies are bound to offer impact to health care for people of the Third World. In this short review, the laboratory and successful clinical test, merits and demerits in comparison with the currently used bioreactors, methods of extraction, and cost estimation of transgenic plant products for medical purposes are briefly reviewed.

Ballistosporomyces changbaiensis sp. nov. and Ballistosporomyces bomiensis sp. nov., two novel species isolated from shrub plant leaves.

PubMed

Han, Pei-Jie; Li, Ai-Hua; Wang, Qi-Ming; Bai, Feng-Yan

2016-07-01

Four strains, CB 266(T), CB 272, XZ 44D1(T) and XZ 49D2, isolated from shrub plant leaves in China were identified as two novel species of the genus Ballistosporomyces by the sequence analysis of the small subunit of ribosomal RNA (SSU rRNA), the D1/D2 domains of the large subunit of rRNA (LSU rRNA) and internal transcribed spacer (ITS) + 5.8S rRNA region, and physiological comparisons. Ballistosporomyces changbaiensis sp. nov. (type strain CB 266(T) = CGMCC 2.02298(T) = CBS 10124(T), Mycobank number MB 815700) and Ballistosporomyces bomiensis sp. nov. (type strain XZ 44D1(T) = CGMCC 2.02661(T) = CBS 12512(T), Mycobank number MB 815701) are proposed to accommodate these two new species.

Molecular cloning and characterization of the α-glucosidase II from Bombyx mori and Spodoptera frugiperda.

PubMed

Watanabe, Satoko; Kakudo, Akemi; Ohta, Masato; Mita, Kazuei; Fujiyama, Kazuhito; Inumaru, Shigeki

2013-04-01

The α-glucosidase II (GII) is a heterodimer of α- and β-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of α-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the α-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII α- and β-subunit genes and the GII α-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII α-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl α-d-glucopyranoside (pNP-αGlc) but were inactive toward N-glycan. Although the B. mori GII β-subunit was not required for the hydrolysis of pNP-αGlc, a B. mori GII complex of the α- and β-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII α-subunit protein was less sensitive to α-glucosidase inhibitors than was the S. frugiperda GII α-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of α-glucosidase inhibitors from mulberry leaves. Copyright © 2013 Elsevier Ltd. All rights reserved.

Agonist-induced Ca2+ Sensitization in Smooth Muscle

PubMed Central

Artamonov, Mykhaylo V.; Momotani, Ko; Stevenson, Andra; Trentham, David R.; Derewenda, Urszula; Derewenda, Zygmunt S.; Read, Paul W.; Gutkind, J. Silvio; Somlyo, Avril V.

2013-01-01

Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca2+]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca2+ sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca2+-sensitized force are not well understood. Using permeabilized blood vessels from PRG(−/−) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca2+-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5′-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca2+ transient and phasic force components and the onset of Ca2+-sensitized force. PMID:24106280

Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains

PubMed Central

2013-01-01

Background Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized. Results In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity. Conclusion Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin. PMID:23856002

Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains.

PubMed

Defraia, Christopher T; Wang, Yongsheng; Yao, Jiqiang; Mou, Zhonglin

2013-07-16

Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized. In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity. Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin.

Radiosensitizing effect of PSMC5, a 19S proteasome ATPase, in H460 lung cancer cells

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

Yim, Ji-Hye; Yun, Hong Shik; Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791

2016-01-01

The function of PSMC5 (proteasome 26S subunit, ATPase 5) in tumors, particularly with respect to cancer radioresistance, is not known. Here, we identified PSMC5 as a novel radiosensitivity biomarker, demonstrating that radiosensitive H460 cells were converted to a radioresistance phenotype by PSMC5 depletion. Exposure of H460 cells to radiation induced a marked accumulation of cell death-promoting reactive oxygen species, but this effect was blocked in radiation-treated H460 PSMC5-knockdown cells through downregulation of the p53-p21 pathway. Interestingly, PSMC5 depletion in H460 cells enhanced both AKT activation and MDM2 transcription, thereby promoting the degradation of p53 and p21 proteins. Furthermore, specific inhibitionmore » of AKT with triciribine or knockdown of MDM2 with small interfering RNA largely restored p21 expression in PSMC5-knockdown H460 cells. Our data suggest that PSMC5 facilitates the damaging effects of radiation in radiation-responsive H460 cancer cells and therefore may serve as a prognostic indicator for radiotherapy and molecular targeted therapy in lung cancer patients. - Highlights: • PSMC5 is a radiation-sensitive biomarker in H460 cells. • PSMC5 depletion inhibits radiation-induced apoptosis in H460 cells. • PSMC5 knockdown blocks ROS generation through inhibition of the p53-p21 pathway. • PSMC5 knockdown enhances p21 degradation via AKT-dependent MDM2 stabilization.« less

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae).

PubMed

Weitemier, Kevin; Straub, Shannon C K; Fishbein, Mark; Liston, Aaron

2015-01-01

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual's consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the "noncoding" ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming).

Changes in growth, carbon and nitrogen enzyme activity and mRNA accumulation in the halophilic microalga Dunaliella viridis in response to NaCl stress

NASA Astrophysics Data System (ADS)

Wang, Dongmei; Wang, Weiwei; Xu, Nianjun; Sun, Xue

2016-12-01

Many species of microalga Dunaliella exhibit a remarkable tolerance to salinity and are therefore ideal for probing the effects of salinity. In this work, we assessed the effects of NaCl stress on the growth, activity and mRNA level of carbon and nitrogen metabolism enzymes of D. viridis. The alga could grow over a salinity range of 0.44 mol L-1 to 3.00 mol L-1 NaCl, but the most rapid growth was observed at 1.00 mol L-1 NaCl, followed by 2.00 mol L-1 NaCl. Paralleling these growth patterns, the highest initial and total Rubisco activities were detected in the presence of 1.00 mol L-1 NaCl, decreasing to 37.33% and 26.39% of those values, respectively, in the presence of 3.00 mol L-1 NaCl, respectively. However, the highest extracellular carbonic anhydrase (CA) activity was measured in the presence of 2.00 mol L-1 NaCl, followed by 1.00 mol L-1 NaCl. Different from the two carbon enzymes, nitrate reductase (NR) activity showed a slight change under different NaCl concentrations. At the transcriptional level, the mRNAs of Rubisco large subunit ( rbcL), and small subunit ( rbcS), attained their highest abundances in the presence of 1.00 and 2.00 mol L-1 NaCl, respectively. The CA mRNA accumulation was induced from 0.44 mol L-1 to 3.00 mol L-1 NaCl, but the NR mRNA showed the decreasing tendency with the increasing salinity. In conclusion, the growth and carbon fixation enzyme of Rubisco displayed similar tendency in response to NaCl stress, CA was proved be salt-inducible within a certain salinity range and NR showed the least effect by NaCl in D. viridis.

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.

PubMed

Duperray, A; Berthier, R; Chagnon, E; Ryckewaert, J J; Ginsberg, M; Plow, E; Marguerie, G

1987-06-01

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.

Interdependence of Pes1, Bop1, and WDR12 controls nucleolar localization and assembly of the PeBoW complex required for maturation of the 60S ribosomal subunit.

PubMed

Rohrmoser, Michaela; Hölzel, Michael; Grimm, Thomas; Malamoussi, Anastassia; Harasim, Thomas; Orban, Mathias; Pfisterer, Iris; Gruber-Eber, Anita; Kremmer, Elisabeth; Eick, Dirk

2007-05-01

The PeBoW complex is essential for cell proliferation and maturation of the large ribosomal subunit in mammalian cells. Here we examined the role of PeBoW-specific proteins Pes1, Bop1, and WDR12 in complex assembly and stability, nucleolar transport, and pre-ribosome association. Recombinant expression of the three subunits is sufficient for complex formation. The stability of all three subunits strongly increases upon incorporation into the complex. Only overexpression of Bop1 inhibits cell proliferation and rRNA processing, and its negative effects could be rescued by coexpression of WDR12, but not Pes1. Elevated levels of Bop1 induce Bop1/WDR12 and Bop1/Pes1 subcomplexes. Knockdown of Bop1 abolishes the copurification of Pes1 with WDR12, demonstrating Bop1 as the integral component of the complex. Overexpressed Bop1 substitutes for endogenous Bop1 in PeBoW complex assembly, leading to the instability of endogenous Bop1. Finally, indirect immunofluorescence, cell fractionation, and sucrose gradient centrifugation experiments indicate that transport of Bop1 from the cytoplasm to the nucleolus is Pes1 dependent, while Pes1 can migrate to the nucleolus and bind to preribosomal particles independently of Bop1. We conclude that the assembly and integrity of the PeBoW complex are highly sensitive to changes in Bop1 protein levels.

Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

PubMed Central

Chiaruttini, C; Expert-Bezançon, A; Hayes, D; Ehresmann, B

1982-01-01

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide. Images PMID:6760129

1H-cyclopentapyrimidine-2,4(1H,3H)-dione-related ionotropic glutamate receptors ligands. structure-activity relationships and identification of potent and Selective iGluR5 modulators.

PubMed

Butini, Stefania; Pickering, Darryl S; Morelli, Elena; Coccone, Salvatore Sanna; Trotta, Francesco; De Angelis, Meri; Guarino, Egeria; Fiorini, Isabella; Campiani, Giuseppe; Novellino, Ettore; Schousboe, Arne; Christensen, Jeppe K; Gemma, Sandra

2008-10-23

(S)-CPW399 ((S)-1) is a potent and excitotoxic AMPA receptor partial agonist. Modifying the cyclopentane ring of (S)-1, we developed two of the most potent and selective functional antagonists (5 and 7) for kainate receptor (KA-R) subunit iGluR5. Derivatives 5 and 7, with their unique pharmacological profile, may lead to a better understanding of the different roles and modes of action of iGluR1-5 subunits, paving the way for the synthesis of new potent, subunit selective iGluR5 modulators.

A monograph of the entomopathogenic genera Hypocrella, Moelleriella, and Samuelsia gen. nov. (Ascomycota, Hypocreales, Clavicipitaceae), and their aschersonia-like anamorphs in the Neotropics

PubMed Central

Chaverri, P.; Liu, M.; Hodge, K.T.

2008-01-01

The present taxonomic revision deals with Neotropical species of three entomopathogenic genera that were once included in Hypocrella s. l.: Hypocrella s. str. (anamorph Aschersonia), Moelleriella (anamorph aschersonia-like), and Samuelsia gen. nov (anamorph aschersonia-like). Species of Hypocrella, Moelleriella, and Samuelsia are pathogens of scale insects (Coccidae and Lecaniidae, Homoptera) and whiteflies (Aleyrodidae, Homoptera) and are common in tropical regions. Phylogenetic analyses of DNA sequences from nuclear ribosomal large subunit (28S), translation elongation factor 1-α (TEF 1-α), and RNA polymerase II subunit 1 (RPB1) and analyses of multiple morphological characters demonstrate that the three segregated genera can be distinguished by the disarticulation of the ascospores and shape and size of conidia. Moelleriella has filiform multi-septate ascospores that disarticulate at the septa within the ascus and aschersonia-like anamorphs with fusoid conidia. Hypocrella s. str. has filiform to long-fusiform ascospores that do not disarticulate and Aschersonia s. str. anamorphs with fusoid conidia. The new genus proposed here, Samuelsia, has filiform to long-fusiform ascospores that do not disarticulate and aschersonia-like anamorphs with small allantoid conidia. In addition, the present study presents and discusses the evolution of species, morphology, and ecology in Hypocrella, Moelleriella, and Samuelsia based on multigene phylogenetic analyses. PMID:18490956

Spectroscopic insights into the oxygen-tolerant membrane-associated [NiFe] hydrogenase of Ralstonia eutropha H16.

PubMed

Saggu, Miguel; Zebger, Ingo; Ludwig, Marcus; Lenz, Oliver; Friedrich, Bärbel; Hildebrandt, Peter; Lendzian, Friedhelm

2009-06-12

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.

Secondary Structure and Subunit Composition of Soy Protein In Vitro Digested by Pepsin and Its Relation with Digestibility

PubMed Central

Yang, Yong; Wang, Zhongjiang; Wang, Rui; Sui, Xiaonan; Qi, Baokun; Han, Feifei; Li, Yang; Jiang, Lianzhou

2016-01-01

In the present study, in vitro digestibility and structure of soybean protein isolates (SPIs) prepared from five soybean varieties were investigated in simulated gastric fluid (SGF), using FT-IR microspectroscopy and SDS-PAGE. The result indicated that β-conformations were prone to be hydrolyzed by pepsin preferentially and transformed to unordered structure during in vitro digestion, followed by the digestion of α-helix and unordered structure. A negative linear correlation coefficient was found between the β-conformation contents of five SPIs and their in vitro digestibility values. The intensities of the protein bands corresponding to 7S and 11S fractions were decreased and many peptide bands appeared at 11~15 kDa during enzymatic hydrolysis. β-conglycinin was poorly hydrolyzed with pepsin, especially the β-7S subunit. On the other hand, basic polypeptides of glycinin degraded slower than acidic polypeptides and represented a large proportion of the residual protein after digestion. 11S-A3 of all SPIs disappeared after 1 h digestion. Moreover, a significant negative linear correlation coefficient (r = −0.89) was found between the β-7S contents of five SPIs and their in vitro digestibility values. These results are useful for further studies of the functional properties and bioactive properties of these varieties and laid theoretical foundations for the development of the specific functional soy protein isolate. PMID:27298825

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

PubMed

Peng, X; Katz, M; Gerzanich, V; Anand, R; Lindstrom, J

1994-03-01

The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

The Purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features

PubMed Central

Derrien, Benoît; Majeran, Wojciech; Effantin, Grégory; Ebenezer, Joseph; Friso, Giulia; van Wijk, Klaas J.; Steven, Alasdair C.; Maurizi, Michael R.; Vallon, Olivier

2012-01-01

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits PMID:22772861

Splicing Factor 2-Associated Protein p32 Participates in Ribosome Biogenesis by Regulating the Binding of Nop52 and Fibrillarin to Preribosome Particles*

PubMed Central

Yoshikawa, Harunori; Komatsu, Wataru; Hayano, Toshiya; Miura, Yutaka; Homma, Keiichi; Izumikawa, Keiichi; Ishikawa, Hideaki; Miyazawa, Naoki; Tachikawa, Hiroyuki; Yamauchi, Yoshio; Isobe, Toshiaki; Takahashi, Nobuhiro

2011-01-01

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52. PMID:21536856

Multilocus sequence analysis of Echinococcus granulosus strains isolated from humans and animals in Iran.

PubMed

Nikmanesh, Bahram; Mirhendi, Hossein; Mahmoudi, Shahram; Rokni, Mohammad Bagher

2017-12-01

Echinococcus granulosus is now considered a complex consisting of at least four species and ten genotypes. Different molecular targets have been described for molecular characterization of E. granulosus; however, in almost all studies only one or two of the targets have been used, and only limited data is available on the utilization of multiple loci. Therefore, we investigated the genetic diversity among 64 strains isolated from 138 cyst specimens of human and animal isolates, using a set of nuclear and mitochondrial genes; i.e., cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 1 (nad1), ATPase subunit 6 (atp6), 12S rRNA (12S), and Actin II (act II). In comparison to the use of molecular reference targets (nad1 + cox1), using singular target (act II or 12S or atp6) yielded lower discriminatory power. Act II and 12S genes could accurately discriminate the G6 genotype, but they were not able to differentiate between G1 and G3 genotypes. As the G1 and G3 genotypes belong to the E. granulosus sensu stricto, low intra-species variation was observed for act II and 12S. The atp6 gene could identify the G3 genotype but could not differentiate G6 and G1 genotypes. Using concatenated sequence of five genes (cox1 + nad1 + atp6 + 12S + act II), genotypes were identified accurately, and markedly higher resolution was obtained in comparison with the use of reference markers (nad1 + cox1) only. Application of multilocus sequence analysis (MLSA) to large-scale studies could provide valuable epidemiological data to make efficient control and management measures for cystic echinococcosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

PubMed Central

Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P.; Romby, Pascale; Springer, Mathias; Marzi, Stefano

2013-01-01

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain.

PubMed

Shi, Hang; Rojas, Raul; Bonifacino, Juan S; Hurley, James H

2006-06-01

The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29 and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1-A resolution reveals two curved beta-sandwich domains connected by a polar core and a flexible linker. Vps26 has an unpredicted structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a glycine in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.

RLE (Research Laboratory of Electronics) Progress Report Number 126.

DTIC Science & Technology

1984-01-01

Loudness 184 26.3 Binaural Hearing 186 S.26.4 Hearing Aid Research 188 26.5 Discrimination of Spectral Shape 191 26.6 Tactile Perception of Speech... beating in the pulse. It is these high intensities which are responsible for large A.C. Stark shifts and ionization RLE P.R. No. 126 12 * . . . Atomic...Department of Aeronautics and Astronautics, Massachusetts Institute of Technology, 1984. 26.3 Binaural Hearing National Institutes of Health (Grant

Pharmacological and immunochemical characterization of α2* nicotinic acetylcholine receptors (nAChRs) in mouse brain

PubMed Central

Whiteaker, Paul; Wilking, Jennifer A; Brown, Robert WB; Brennan, Robert J; Collins, Allan C; Lindstrom, Jon M; Boulter, Jim

2009-01-01

Aim: α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2* nAChRs in these mouse brain regions. Methods: α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing (α2*) nAChRs in these two regions. Results: [125I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8±1.7 and 92±26 fmol̇mg-1 protein, respectively). Pharmacological characterization using the β2-selective drug A85380 to inhibit [125I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2* nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good (>90% of β2* and >80% of β4* nAChRs were routinely recovered). Conclusion: Immunoprecipitation experiments indicated that interpeduncular nucleus α2* nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits. PMID:19498420

Mediator kinase module and human tumorigenesis.

PubMed

Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

2015-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

Mediator kinase module and human tumorigenesis

PubMed Central

Clark, Alison D.; Oldenbroek, Marieke; Boyer, Thomas G.

2016-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit “kinase” module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways. PMID:26182352

hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0

PubMed Central

Tomecki, Rafal; Labno, Anna; Drazkowska, Karolina; Cysewski, Dominik; Dziembowski, Andrzej

2015-01-01

Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5′-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site. PMID:26237581

Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: evidence for a novel site of streptomycin resistance in the small subunit rRNA.

PubMed

Gauthier, A; Turmel, M; Lemieux, C

1988-10-01

A major obstacle to our understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.

Dissociation of Multisubunit Protein-Ligand Complexes in the Gas Phase. Evidence for Ligand Migration

NASA Astrophysics Data System (ADS)

Zhang, Yixuan; Deng, Lu; Kitova, Elena N.; Klassen, John S.

2013-10-01

The results of collision-induced dissociation (CID) experiments performed on gaseous protonated and deprotonated ions of complexes of cholera toxin B subunit homopentamer (CTB5) with the pentasaccharide (β-D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal p-(1→4)-β-D-Glc p (GM1)) and corresponding glycosphingolipid (β-D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal p-(1→4)-β-D-Glc p-Cer (GM1-Cer)) ligands, and the homotetramer streptavidin (S4) with biotin (B) and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N-(biotinyl) (Btl), are reported. The protonated (CTB5 + 5GM1)n+ ions dissociated predominantly by the loss of a single subunit, with the concomitant migration of ligand to another subunit. The simultaneous loss of ligand and subunit was observed as a minor pathway. In contrast, the deprotonated (CTB5 + 5GM1)n- ions dissociated preferentially by the loss of deprotonated ligand; the loss of ligand-bound and ligand-free subunit were minor pathways. The presence of ceramide (Cer) promoted ligand migration and the loss of subunit. The main dissociation pathway for the protonated and deprotonated (S4 + 4B)n+/- ions, as well as for deprotonated (S4 + 4Btl)n- ions, was loss of the ligand. However, subunit loss from the (S4 + 4B)n+ ions was observed as a minor pathway. The (S4 + 4Btl)n+ ions dissociated predominantly by the loss of free and ligand-bound subunit. The charge state of the complex and the collision energy were found to have little effect on the relative contribution of the different dissociation channels. Thermally-driven ligand migration between subunits was captured in the results of molecular dynamics simulations performed on protonated (CTB5 + 5GM1)15+ ions (with a range of charge configurations) at 800 K. Notably, the migration pathway was found to be highly dependent on the charge configuration of the ion. The main conclusion of this study is that the dissociation pathways of multisubunit protein-ligand complexes in the gas phase depend, not only on the native topology of the complex, but also on structural changes that occur upon collisional activation.

THE MITOTIC APPARATUS

PubMed Central

Stephens, R. E.

1967-01-01

The major 22S protein of the hexylene glycol-isolated mitotic apparatus has been characterized from spindle isolates and extracts of whole eggs and acetone powders of eggs from the sea urchins Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Arbacia punctulata. The protein is free of nucleotide, lipid, and ATPase activity. Essentially identical in amino acid composition, proteins from these species show a relatively high content of glutamic and aspartic acids and are fairly rich in hydrophobic amino acids. Optical rotatory dispersion studies indicate a helical content of about 20%, a value consistent with the proline content of the protein. The purified proteins have sedimentation rates in the range of 22–24S, diffusion constants of 2.4–2.5F, intrinsic viscosities of 3.7–4.3 ml/g, a partial specific volume of 0.74, and an average molecular weight of 880,000. Electron microscopy indicates a globular molecule with dimensions of approximately 150 by 200 A; such size and symmetry are consistent with hydrodynamic measurements. The 22S protein yields 6–7S, 9–10S, and 13–14S subunits below pH 4 or above pH 11. The 13–14S component has an estimated molecular weight of 600,000–700,000. A 5–6S particle is formed in 8 M urea or 5 M guanidine hydrochloride, while at pH 12 the 6–7S subunit is seen; each particle has a molecular weight of 230,000–240,000. In 8 M urea plus 2% mercaptoethanol or at pH 13, the molecular weight becomes 105,000–120,000; under these conditions the particle sediments at 2.5–3S and 4S, respectively. On the basis of these molecular weights, the 6–7S, 9–10S, 13–14S, and the parent 22S particle should be dimer, tetramer, hexamer, and octamer, respectively, of the 105,000–120,000 molecular weight subunit. The various subunits will reform the 22S particle when returned to neutral buffer, with the exception of the mercaptoethanol-treated urea subunit where breakage of disulfide bonds results in a polydisperse aggregate. The 22S particle itself is not susceptible to sulfhydryl reagents, implying either that the disulfide bonds are inaccessible or that they are unnecessary for maintenance of tertiary structure once the 22S particle has formed from subunits. PMID:10976220

Tuning the charge carrier density in the thermoelectric colusite

NASA Astrophysics Data System (ADS)

Kim, Fiseong S.; Suekuni, Koichiro; Nishiate, Hirotaka; Ohta, Michihiro; Tanaka, Hiromi I.; Takabatake, Toshiro

2016-05-01

The colusite Cu26V2Sn6S32 has high potential as a thermoelectric material at medium-high temperatures because of a large Seebeck coefficient (S ≃ 220 μV/K) and rather small electrical resistivity (ρ ≃ 100 μΩm) at 660 K. To improve the thermoelectric performance, we have tuned the hole carrier density p by substituting Zn for Cu in Cu26-xZnxV2Sn6S32 (x = 1-3) and starting with Cu and Sn deficient compositions in Cu26-yV2Sn6S32 (y = 1, 2) and Cu26V2Sn6-zS32 (z = 0.25-1), respectively. Powder x-ray diffraction and electron-probe microanalysis showed that the Zn-substituted samples and Sn-deficient (z ≥ 0.5) samples are formed in a single phase, whereas the Cu26-yV2Sn6S32 samples are composed of two phases with slightly different compositions. Within these samples, the value of p at 300 K varies in the range between 3.6 × 1020 and 2.8 × 1021 cm-3. The relation between p and S led to the effective mass m* of 4-7m0 for the hole carriers. The large S of the colusite is therefore ascribed to the heavy mass carriers of the valence band top. The decreases in p with x and y reduced the dimensionless thermoelectric figure of merit ZT, whereas the increase in p with z raised ZT from 0.56 (z = 0) to 0.62 (z = 0.5) at 660 K.

Conopeptide Vt3.1 preferentially inhibits BK potassium channels containing β4 subunits via electrostatic interactions.

PubMed

Li, Min; Chang, Shan; Yang, Longjin; Shi, Jingyi; McFarland, Kelli; Yang, Xiao; Moller, Alyssa; Wang, Chunguang; Zou, Xiaoqin; Chi, Chengwu; Cui, Jianmin

2014-02-21

BK channel β subunits (β1-β4) modulate the function of channels formed by slo1 subunits to produce tissue-specific phenotypes. The molecular mechanism of how the homologous β subunits differentially alter BK channel functions and the role of different BK channel functions in various physiologic processes remain unclear. By studying channels expressed in Xenopus laevis oocytes, we show a novel disulfide-cross-linked dimer conopeptide, Vt3.1 that preferentially inhibits BK channels containing the β4 subunit, which is most abundantly expressed in brain and important for neuronal functions. Vt3.1 inhibits the currents by a maximum of 71%, shifts the G-V relation by 45 mV approximately half-saturation concentrations, and alters both open and closed time of single channel activities, indicating that the toxin alters voltage dependence of the channel. Vt3.1 contains basic residues and inhibits voltage-dependent activation by electrostatic interactions with acidic residues in the extracellular loops of the slo1 and β4 subunits. These results suggest a large interaction surface between the slo1 subunit of BK channels and the β4 subunit, providing structural insight into the molecular interactions between slo1 and β4 subunits. The results also suggest that Vt3.1 is an excellent tool for studying β subunit modulation of BK channels and for understanding the physiological roles of BK channels in neurophysiology.

[Three regions of Rpb10 mini-subunit of nuclear RNA polymerases are strictly conserved in all eukaryotes].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N

1996-12-01

The rpb10+ cDNA from the fission yeast Schizosaccharomyces pombe was cloned using two independent approaches (PCR and genetic suppression). The cloned cDNA encoded the Rpb10 subunit common for all three RNA polymerases. Comparison of the deduced amino acid sequence of the Sz. pombe Rbp10 subunit (71 amino acid residues) with those of the homologous subunits of RNA polymerases I, II, and III from Saccharomyces cerevisiae and Home sapiens revealed that heptapeptides RCFT/SCGK (residues 6-12), RYCCRRM (residues 43-49), and HVDLIEK (residues 53-59) were evolutionarily the most conserved structural motifs of these subunits. It is shown that the Rbp10 subunit from Sz. pombe can substitute its homolog (ABC10 beta) in the baker's yeast S. cerevisiae.

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

PubMed Central

Huynh, Nhung T.; Hesketh, Emma L.; Saxena, Pooja; Meshcheriakova, Yulia; Ku, You-Chan; Hoang, Linh T.; Johnson, John E.; Ranson, Neil A.; Lomonossoff, George P.; Reddy, Vijay S.

2016-01-01

SUMMARY Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed. PMID:27021160

Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

PubMed Central

Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.

1998-01-01

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. PMID:9464394

Subunit architecture and functional modular rearrangements of the transcriptional Mediator complex

PubMed Central

Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2014-01-01

SUMMARY The multisubunit Mediator comprising ~30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. PMID:24882805

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

PubMed

Frébortova; Matsushita; Arata; Adachi

1998-01-27

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

A Family of G Protein βγ Subunits Translocate Reversibly from the Plasma Membrane to Endomembranes on Receptor Activation*S

PubMed Central

Saini, Deepak Kumar; Kalyanaraman, Vani; Chisari, Mariangela; Gautam, Narasimhan

2008-01-01

The present model of G protein activation by G protein-coupled receptors exclusively localizes their activation and function to the plasma membrane (PM). Observation of the spatiotemporal response of G protein subunits in a living cell to receptor activation showed that 6 of the 12 members of the G protein γ subunit family translocate specifically from the PM to endomembranes. The γ subunits translocate as βγ complexes, whereas the α subunit is retained on the PM. Depending on the γ subunit, translocation occurs predominantly to the Golgi complex or the endoplasmic reticulum. The rate of translocation also varies with the γ subunit type. Different γ subunits, thus, confer distinct spatiotemporal properties to translocation. A striking relationship exists between the amino acid sequences of various γ subunits and their translocation properties. γ subunits with similar translocation properties are more closely related to each other. Consistent with this relationship, introducing residues conserved in translocating subunits into a non-translocating subunit results in a gain of function. Inhibitors of vesicle-mediated trafficking and palmitoylation suggest that translocation is diffusion-mediated and controlled by acylation similar to the shuttling of G protein subunits (Chisari, M., Saini, D. K., Kalyanaraman, V., and Gautam, N. (2007) J. Biol. Chem. 282, 24092–24098). These results suggest that the continual testing of cytosolic surfaces of cell membranes by G protein subunits facilitates an activated cell surface receptor to direct potentially active G protein βγ subunits to intracellular membranes. PMID:17581822

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop.

PubMed

Potter, J W; Black, C C

1982-08-01

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo(35)S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the (35)S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

Assembling a protein-protein interaction map of the SSU processome from existing datasets.

PubMed

Lim, Young H; Charette, J Michael; Baserga, Susan J

2011-03-10

The small subunit (SSU) processome is a large ribonucleoprotein complex involved in small ribosomal subunit assembly. It consists of the U3 snoRNA and ∼72 proteins. While most of its components have been identified, the protein-protein interactions (PPIs) among them remain largely unknown, and thus the assembly, architecture and function of the SSU processome remains unclear. We queried PPI databases for SSU processome proteins to quantify the degree to which the three genome-wide high-throughput yeast two-hybrid (HT-Y2H) studies, the genome-wide protein fragment complementation assay (PCA) and the literature-curated (LC) datasets cover the SSU processome interactome. We find that coverage of the SSU processome PPI network is remarkably sparse. Two of the three HT-Y2H studies each account for four and six PPIs between only six of the 72 proteins, while the third study accounts for as little as one PPI and two proteins. The PCA dataset has the highest coverage among the genome-wide studies with 27 PPIs between 25 proteins. The LC dataset was the most extensive, accounting for 34 proteins and 38 PPIs, many of which were validated by independent methods, thereby further increasing their reliability. When the collected data were merged, we found that at least 70% of the predicted PPIs have yet to be determined and 26 proteins (36%) have no known partners. Since the SSU processome is conserved in all Eukaryotes, we also queried HT-Y2H datasets from six additional model organisms, but only four orthologues and three previously known interologous interactions were found. This provides a starting point for further work on SSU processome assembly, and spotlights the need for a more complete genome-wide Y2H analysis.

Assembling a Protein-Protein Interaction Map of the SSU Processome from Existing Datasets

PubMed Central

Baserga, Susan J.

2011-01-01

Background The small subunit (SSU) processome is a large ribonucleoprotein complex involved in small ribosomal subunit assembly. It consists of the U3 snoRNA and ∼72 proteins. While most of its components have been identified, the protein-protein interactions (PPIs) among them remain largely unknown, and thus the assembly, architecture and function of the SSU processome remains unclear. Methodology We queried PPI databases for SSU processome proteins to quantify the degree to which the three genome-wide high-throughput yeast two-hybrid (HT-Y2H) studies, the genome-wide protein fragment complementation assay (PCA) and the literature-curated (LC) datasets cover the SSU processome interactome. Conclusions We find that coverage of the SSU processome PPI network is remarkably sparse. Two of the three HT-Y2H studies each account for four and six PPIs between only six of the 72 proteins, while the third study accounts for as little as one PPI and two proteins. The PCA dataset has the highest coverage among the genome-wide studies with 27 PPIs between 25 proteins. The LC dataset was the most extensive, accounting for 34 proteins and 38 PPIs, many of which were validated by independent methods, thereby further increasing their reliability. When the collected data were merged, we found that at least 70% of the predicted PPIs have yet to be determined and 26 proteins (36%) have no known partners. Since the SSU processome is conserved in all Eukaryotes, we also queried HT-Y2H datasets from six additional model organisms, but only four orthologues and three previously known interologous interactions were found. This provides a starting point for further work on SSU processome assembly, and spotlights the need for a more complete genome-wide Y2H analysis. PMID:21423703

Structure of EvaA: a paradigm for sugar 2,3-dehydratases.

PubMed

Kubiak, Rachel L; Thoden, James B; Holden, Hazel M

2013-03-26

Unusual deoxysugars found appended to natural products often provide or enhance the pharmacokinetic activities of the parent compound. The preferred carbohydrate donors for the biosynthesis of such glycosylated natural products are the dTDP-linked sugars. Many of the biologically relevant dTDP-deoxysugars are constructed around the 2,6-dideoxyhexoses or the 2,3(4),6-trideoxyhexoses. A key step in the biosynthesis of these sugars is the removal of the hexose C-2' hydroxyl group and the oxidation of the C-3' hydroxyl group to a carbonyl moiety. Enzymes that catalyze these reactions are referred to as 2,3-dehydratases and have been, for the most part, largely uncharacterized. Here we report the first structural analysis of a sugar 2,3-dehydratase. For this investigation, the enzyme, EvaA, was cloned from Amycolatopsis orientalis, and the structure was solved and refined to a nominal resolution of 1.7 Å. On the basis of the resulting model, it is clear that EvaA belongs to the large Nudix hydrolase superfamily and is most similar to GDP-mannose hydrolase. Each subunit of the EvaA dimer folds into two domains that clearly arose via gene duplication. Two dTDP-sugar binding pockets, A and B, are present in each EvaA subunit. On the basis of site-directed mutagenesis experiments and activity assays, it appears that pocket A functions as the active site and pocket B is simply a remnant left behind from the gene duplication event. As 2,3-dehydration is crucial for the biosynthesis of many unusual deoxysugars, this investigation provides key structural insight into this widely conserved reaction.

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

PubMed Central

Ghosh, D; Weeks, C M; Grochulski, P; Duax, W L; Erman, M; Rimsay, R L; Orr, J C

1991-01-01

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. Images PMID:1946424

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

PubMed

Espinar-Marchena, Francisco; Rodríguez-Galán, Olga; Fernández-Fernández, José; Linnemann, Jan; de la Cruz, Jesús

2018-05-18

The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits.

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

PubMed Central

Espinar-Marchena, Francisco; Rodríguez-Galán, Olga; Fernández-Fernández, José; Linnemann, Jan; de la Cruz, Jesús

2018-01-01

Abstract The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits. PMID:29788267

The structure of subunit E of the Pyrococcus horikoshii OT3 A-ATP synthase gives insight into the elasticity of the peripheral stalk.

PubMed

Balakrishna, Asha Manikkoth; Hunke, Cornelia; Grüber, Gerhard

2012-07-13

A(1)A(O) ATP synthases are the major energy converters of archaea. They are composed of an A(1) region that synthesizes ATP and an integral part A(O) that conducts ions. Subunit E is a component of the peripheral stalk that links the A(1) with the A(O) part of the A-ATP synthase. We have determined the crystal structure of the entire subunit E (PhE) of the Pyrococcus horikoshii OT3 A-ATP synthase at 3.6 Å resolution. The structure reveals an extended S-shaped N-terminal α-helix with 112.29 Å in length, followed by a globular head group. The S-shaped feature, common in elastic connectors and spacers, would facilitate the storage of transient elastic energy during rotary motion in the enzyme. The structure has been superimposed into the asymmetric peripheral stalks of the three-dimensional reconstruction of the Pyrococcus furiosus enzyme, revealing that the S-shaped subunit PhE fits well into the bent peripheral stalk, whereas the previously solved E subunit structure (3.1 Å resolution) of Thermus thermophilus A-ATP synthase is well accommodated in the density of the straight stator domain. The different features of the two stalk subunits are discussed in light of a novel coupling mechanism in A-ATP synthases proposed to differ from the Wankel engine of F-ATP synthases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus , Sulfolobus tokodaii and Methanocaldococcus jannaschii

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

Watanabe, Yuzo; Yanai, Hisaaki; Kanagawa, Mayumi

2016-07-27

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, fromThermus thermophilus,Sulfolobus tokodaiiandMethanocaldococcus jannaschiiwere determined and their structural characteristics were analyzed. For PurS fromT. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations ofmore » the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.« less

Intradermal Vaccination With Adjuvanted Ebola Virus Soluble Glycoprotein Subunit Vaccine by Microneedle Patches Protects Mice Against Lethal Ebola Virus Challenge.

PubMed

Liu, Ying; Ye, Ling; Lin, Fang; Gomaa, Yasmine; Flyer, David; Carrion, Ricardo; Patterson, Jean L; Prausnitz, Mark R; Smith, Gale; Glenn, Gregory; Wu, Hua; Compans, Richard W; Yang, Chinglai

2018-06-08

In this study, we investigated immune responses induced by purified Ebola virus (EBOV) soluble glycoprotein (sGP) subunit vaccines via intradermal immunization with microneedle (MN) patches in comparison with intramuscular (IM) injection in mice. Our results showed that MN delivery of EBOV sGP was superior to IM injection in eliciting higher levels and longer lasting antibody responses against EBOV sGP and GP antigens. Moreover, sGP-specific immune responses induced by MN or IM immunizations were effectively augmented by formulating sGP with a saponin-based adjuvant, and they were shown to confer complete protection of mice against lethal mouse-adapted EBOV (MA-EBOV) challenge. In comparison, mice that received sGP without adjuvant by MN or IM immunizations succumbed to lethal MA-EBOV challenge. These results show that immunization with EBOV sGP subunit vaccines with adjuvant by MN patches, which have been shown to provide improved safety and thermal stability, is a promising approach to protect against EBOV infection.

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

PubMed Central

Pánek, Josef; Kolář, Michal; Vohradský, Jiří; Shivaya Valášek, Leoš

2013-01-01

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs. PMID:23804757

Exploiting hidden symmetry in natural products: total syntheses of amphidinolides C and F.

PubMed

Mahapatra, Subham; Carter, Rich G

2013-07-24

The total synthesis of amphidinolide C and a second-generation synthesis of amphidinolide F have been accomplished through the use of a common intermediate to access both the C1-C8 and the C18-C25 sections. The development of a Ag-catalyzed cyclization of a propargyl benzoate diol is described to access both trans-tetrahydrofuran rings. The evolution of a Felkin-controlled, 2-lithio-1,3-dienyl addition strategy to incorporate C9-C11 diene as well as C8 stereocenter is detailed. Key controlling aspects in the sulfone alkylation/oxidative desulfurization to join the major subunits, including the exploration of the optimum masking group for the C18 carbonyl motif, are discussed. A Trost asymmetric alkynylation and a stereoselective cuprate addition to an alkynoate have been developed for the rapid construction of the C26-C34 subunit. A Tamura/Vedejs olefination to introduce the C26 side arm of amphidnolides C and F is employed. The late-stage incorporation of the C15, C18 diketone motif proved critical to the successful competition of the total syntheses.

Exploiting Hidden Symmetry in Natural Products: Total Syntheses of Amphidinolides C and F

PubMed Central

Mahapatra, Subham

2013-01-01

The total synthesis of amphidinolide C and a second-generation synthesis of amphidinolide F have been accomplished through the use of a common intermediate to access both the C1-C8 and the C18-C25 sections. The development of a Ag-catalyzed cyclization of a propargyl benzoate diol is described to access both trans-tetrahydrofuran rings. The evolution of a Felkin-controlled 2-lithio-1,3-dienyl addition strategy to incorporate C9-C11 diene as well as C8 stereocenter is detailed. Key controlling aspects in the sulfone alkylation / oxidative desulfurization to join the major subunits, including the exploration of the optimum masking group for the C18 carbonyl motif, are discussed. A Trost asymmetric alkynylation and a stereoselective cuprate addition to an alkynoate have been developed for the rapid construction of the C26-C34 subunit. A Tamura/Vedejs olefination to introduce the C26 sidearm of amphidnolides C and F is employed. The late-stage incorporation of the C15, C18 diketone motif proved critical to the successful competition of the total syntheses. PMID:23845005

5SRNAdb: an information resource for 5S ribosomal RNAs.

PubMed

Szymanski, Maciej; Zielezinski, Andrzej; Barciszewski, Jan; Erdmann, Volker A; Karlowski, Wojciech M

2016-01-04

Ribosomal 5S RNA (5S rRNA) is the ubiquitous RNA component found in the large subunit of ribosomes in all known organisms. Due to its small size, abundance and evolutionary conservation 5S rRNA for many years now is used as a model molecule in studies on RNA structure, RNA-protein interactions and molecular phylogeny. 5SRNAdb (http://combio.pl/5srnadb/) is the first database that provides a high quality reference set of ribosomal 5S RNAs (5S rRNA) across three domains of life. Here, we give an overview of new developments in the database and associated web tools since 2002, including updates to database content, curation processes and user web interfaces. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The morphology of cometary dust: Subunit size distributions down to tens of nanometres

NASA Astrophysics Data System (ADS)

Mannel, Thurid; Bentley, Mark; Boakes, Peter; Jeszenszky, Harald; Levasseur-Regourd, Anny-Chantal; Schmied, Roland; Torkar, Klaus

2017-04-01

The Rosetta orbiter carried a dedicated analysis suite for cometary dust. One of the key instruments was MIDAS (Micro-Imaging Dust Analysis System), an atomic force microscope that scanned the surfaces of hundreds of (sub-)micrometre particles in 3D with resolutions down to nanometres. This provided the opportunity to study the morphology of the smallest cometary dust; initial investigation revealed that the particles are agglomerates of smaller subunits [1] with different structural properties [2]. To understand the (surface-) structure of the dust particles and the origin of their smallest building blocks, a number of particles were investigated in detail and the size distribution of their subunits determined [3]. Here we discuss the subunit size distributions ranging from tens of nanometres to a few micrometres. The differences between the subunit size distributions for particles collected pre-perihelion, close to perihelion, and during a huge outburst are examined, as well as the dependence of subunit size on particle size. A case where a particle was fragmented in consecutive scans allows a direct comparison of fragment and subunit size distributions. Finally, the small end of the subunit size distribution is investigated: the smallest determined sizes will be reviewed in the context of other cometary missions, interplanetary dust particles believed to originate from comets, and remote observations. It will be discussed if the smallest subunits can be interpreted as fundamental building blocks of our early Solar System and if their origin was in our protoplanetary disc or the interstellar material. References: [1] M.S. Bentley, R. Schmied, T. Mannel et al., Aggregate dust particles at comet 67P/Chruyumov-Gerasimenko, Nature, 537, 2016. doi:10.1038/nature19091 [2] T. Mannel, M.S. Bentley, R. Schmied et al., Fractal cometary dust - a window into the early Solar system, MNRAS, 462, 2016. doi:10.1093/mnras/stw2898 [3] R. Schmied, T. Mannel, H. Jeszenszky, M.S. Bentley, Properties of cometary dust down to the nanometre scale, poster at the conference 'Comets: A new vision after Rosetta/Philae' in Toulouse, 14-18 November 2016.

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

PubMed Central

Schoch, Conrad L.; Seifert, Keith A.; Huhndorf, Sabine; Robert, Vincent; Spouge, John L.; Levesque, C. André; Chen, Wen; Bolchacova, Elena; Voigt, Kerstin; Crous, Pedro W.; Miller, Andrew N.; Wingfield, Michael J.; Aime, M. Catherine; An, Kwang-Deuk; Bai, Feng-Yan; Barreto, Robert W.; Begerow, Dominik; Bergeron, Marie-Josée; Blackwell, Meredith; Boekhout, Teun; Bogale, Mesfin; Boonyuen, Nattawut; Burgaz, Ana R.; Buyck, Bart; Cai, Lei; Cai, Qing; Cardinali, G.; Chaverri, Priscila; Coppins, Brian J.; Crespo, Ana; Cubas, Paloma; Cummings, Craig; Damm, Ulrike; de Beer, Z. Wilhelm; de Hoog, G. Sybren; Del-Prado, Ruth; Dentinger, Bryn; Diéguez-Uribeondo, Javier; Divakar, Pradeep K.; Douglas, Brian; Dueñas, Margarita; Duong, Tuan A.; Eberhardt, Ursula; Edwards, Joan E.; Elshahed, Mostafa S.; Fliegerova, Katerina; Furtado, Manohar; García, Miguel A.; Ge, Zai-Wei; Griffith, Gareth W.; Griffiths, K.; Groenewald, Johannes Z.; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Guo, Liang-Dong; Hagen, Ferry; Hambleton, Sarah; Hamelin, Richard C.; Hansen, Karen; Harrold, Paul; Heller, Gregory; Herrera, Cesar; Hirayama, Kazuyuki; Hirooka, Yuuri; Ho, Hsiao-Man; Hoffmann, Kerstin; Hofstetter, Valérie; Högnabba, Filip; Hollingsworth, Peter M.; Hong, Seung-Beom; Hosaka, Kentaro; Houbraken, Jos; Hughes, Karen; Huhtinen, Seppo; Hyde, Kevin D.; James, Timothy; Johnson, Eric M.; Johnson, Joan E.; Johnston, Peter R.; Jones, E.B. Gareth; Kelly, Laura J.; Kirk, Paul M.; Knapp, Dániel G.; Kõljalg, Urmas; Kovács, Gábor M.; Kurtzman, Cletus P.; Landvik, Sara; Leavitt, Steven D.; Liggenstoffer, Audra S.; Liimatainen, Kare; Lombard, Lorenzo; Luangsa-ard, J. Jennifer; Lumbsch, H. Thorsten; Maganti, Harinad; Maharachchikumbura, Sajeewa S. N.; Martin, María P.; May, Tom W.; McTaggart, Alistair R.; Methven, Andrew S.; Meyer, Wieland; Moncalvo, Jean-Marc; Mongkolsamrit, Suchada; Nagy, László G.; Nilsson, R. Henrik; Niskanen, Tuula; Nyilasi, Ildikó; Okada, Gen; Okane, Izumi; Olariaga, Ibai; Otte, Jürgen; Papp, Tamás; Park, Duckchul; Petkovits, Tamás; Pino-Bodas, Raquel; Quaedvlieg, William; Raja, Huzefa A.; Redecker, Dirk; Rintoul, Tara L.; Ruibal, Constantino; Sarmiento-Ramírez, Jullie M.; Schmitt, Imke; Schüßler, Arthur; Shearer, Carol; Sotome, Kozue; Stefani, Franck O.P.; Stenroos, Soili; Stielow, Benjamin; Stockinger, Herbert; Suetrong, Satinee; Suh, Sung-Oui; Sung, Gi-Ho; Suzuki, Motofumi; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M. Teresa; Tretter, Eric; Untereiner, Wendy A.; Urbina, Hector; Vágvölgyi, Csaba; Vialle, Agathe; Vu, Thuy Duong; Walther, Grit; Wang, Qi-Ming; Wang, Yan; Weir, Bevan S.; Weiß, Michael; White, Merlin M.; Xu, Jianping; Yahr, Rebecca; Yang, Zhu L.; Yurkov, Andrey; Zamora, Juan-Carlos; Zhang, Ning; Zhuang, Wen-Ying; Schindel, David

2012-01-01

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. PMID:22454494

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.

PubMed

Schoch, Conrad L; Seifert, Keith A; Huhndorf, Sabine; Robert, Vincent; Spouge, John L; Levesque, C André; Chen, Wen

2012-04-17

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets.

PubMed

Bengtsson, Johan; Eriksson, K Martin; Hartmann, Martin; Wang, Zheng; Shenoy, Belle Damodara; Grelet, Gwen-Aëlle; Abarenkov, Kessy; Petri, Anna; Rosenblad, Magnus Alm; Nilsson, R Henrik

2011-10-01

The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa ( http://microbiology.se/software/metaxa/ ), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.

Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells.

PubMed

Tayama, Yoko; Kawahara, Hiroyuki; Minami, Ryosuke; Shimada, Masumi; Yokosawa, Hideyoshi

2007-12-01

The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356-5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.

Data for a proteomic analysis of p53-independent induction of apoptosis by bortezomib

PubMed Central

Yerlikaya, Azmi; Okur, Emrah; Tarık Baykal, Ahmet; Acılan, Ceyda; Boyacı, İhsan; Ulukaya, Engin

2014-01-01

This data article contains data related to the research article entitled, “A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line” by Yerlikaya et al. [1]. The research article presented 2-DE and nLC-MS/MS based proteomic analysis of proteasome inhibitor bortezomib-induced changes in the expression of cellular proteins. The report showed that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24 h. In addition, the report demonstrated that Hsp70, the 26S proteasome non-ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The data here show for the first time the increased expressions of Card10, Dffb, Traf3 and Trp53bp2 in response to inhibition of the 26S proteasome. The information presented here also shows that both Traf1 and Xiap (a member of IAPs) are also downregulated simultaneously upon proteasomal inhibition. The increases in the level of Card10 and Trp53bp2 proteins were verified by Western blot analysis in response to varying concentrations of bortezomib for 24 h. PMID:26217687

Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

NASA Technical Reports Server (NTRS)

Mednieks, M. I.; Popova, I.; Grindeland, R. E.

1992-01-01

Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

Three new anascosporic genera of the Saccharomycotina: Danielozyma gen. nov., Deakozyma gen. nov. and Middelhovenomyces gen. nov.

USDA-ARS?s Scientific Manuscript database

Three new non-ascosporic, ascomycetous yeast genera are proposed based on their isolation from currently described species and genera. Phylogenetic placement of the genera was determined from analysis of nuclear gene sequences for D1/D2 large subunit rRNA, small subunit rRNA, translation elongation...

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

Follis, Kathryn E.; York, Joanne; Nunberg, Jack H.

The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctionalmore » region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.« less

Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

PubMed Central

Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

2016-01-01

Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

KCNE1 constrains the voltage sensor of Kv7.1 K+ channels.

PubMed

Shamgar, Liora; Haitin, Yoni; Yisharel, Ilanit; Malka, Eti; Schottelndreier, Hella; Peretz, Asher; Paas, Yoav; Attali, Bernard

2008-04-09

Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K(+) channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K(+) channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions.

KCNE1 Constrains the Voltage Sensor of Kv7.1 K+ Channels

PubMed Central

Yisharel, Ilanit; Malka, Eti; Schottelndreier, Hella; Peretz, Asher; Paas, Yoav; Attali, Bernard

2008-01-01

Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K+ channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K+ channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions. PMID:18398469

Stoichiometry for binding and transport by the twin arginine translocation system.

PubMed

Celedon, Jose M; Cline, Kenneth

2012-05-14

Twin arginine translocation (Tat) systems transport large folded proteins across sealed membranes. Tat systems accomplish this feat with three membrane components organized in two complexes. In thylakoid membranes, cpTatC and Hcf106 comprise a large receptor complex containing an estimated eight cpTatC-Hcf106 pairs. Protein transport occurs when Tha4 joins the receptor complex as an oligomer of uncertain size that is thought to form the protein-conducting structure. Here, binding analyses with intact membranes or purified complexes indicate that each receptor complex could bind eight precursor proteins. Kinetic analysis of translocation showed that each precursor-bound site was independently functional for transport, and, with sufficient Tha4, all sites were concurrently active for transport. Tha4 titration determined that ∼26 Tha4 protomers were required for transport of each OE17 (oxygen-evolving complex subunit of 17 kD) precursor protein. Our results suggest that, when fully saturated with precursor proteins and Tha4, the Tat translocase is an ∼2.2-megadalton complex that can individually transport eight precursor proteins or cooperatively transport multimeric precursors.

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

Sequence to Structure (S2S): display, manipulate and interconnect RNA data from sequence to structure.

PubMed

Jossinet, Fabrice; Westhof, Eric

2005-08-01

Efficient RNA sequence manipulations (such as multiple alignments) need to be constrained by rules of RNA structure folding. The structural knowledge has increased dramatically in the last years with the accumulation of several large RNA structures similar to those of the bacterial ribosome subunits. However, no tool in the RNA community provides an easy way to link and integrate progress made at the sequence level using the available three-dimensional information. Sequence to Structure (S2S) proposes a framework in which an user can easily display, manipulate and interconnect heterogeneous RNA data, such as multiple sequence alignments, secondary and tertiary structures. S2S has been implemented using the Java language and has been developed and tested under UNIX systems, such as Linux and MacOSX. S2S is available at http://bioinformatics.org/S2S/.

Location of the β4 transmembrane helices in the BK potassium channel

PubMed Central

Wu, Roland S.; Chudasama, Neelesh; Zakharov, Sergey I.; Doshi, Darshan; Motoike, Howard; Liu, Guoxia; Yao, Yongneng; Niu, Xiaowei; Deng, Shi-Xian; Landry, Donald W.; Karlin, Arthur; Marx, Steven O.

2009-01-01

Large-conductance, voltage- and Ca2+-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of α subunits, which contain the voltage-sensor domains and the Ca2+- sensor domains, and form the pore, and often one of four types of β subunits, which modulate the channel in a cell-specific manner. β4 is expressed in neurons throughout the brain. Deletion of β4 in mice causes temporal lobe epilepsy. Compared to channels composed of α alone, channels composed of α and β4 activate and deactivate more slowly. We inferred the locations of the two β4 transmembrane (TM) helices, TM1 and TM2, relative to the seven αTM helices, S0-S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that β4 TM2 is close to α S0 and that β4 TM1 is close to both α S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3 through S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V50 and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states. PMID:19571123

Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II.

PubMed

Dennis, Andrew P; Lonard, David M; Nawaz, Zafar; O'Malley, Bert W

2005-03-01

In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

Biodegradation and detoxification of aliphatic and aromatic hydrocarbons by new yeast strains.

PubMed

Hashem, Mohamed; Alamri, Saad A; Al-Zomyh, Sharefah S A A; Alrumman, Sulaiman A

2018-04-30

Seeking new efficient hydrocarbon-degrading yeast stains was the main goal of this study. Because microorganisms are greatly affected by the environmental factors, the biodegradation potentiality of the microorganisms varies from climatic area to another. This induces research to develop and optimize the endemic organisms in bioremediation technology. In this study, 67 yeast strains were tested for their growth potentiality on both aliphatic and aromatic hydrocarbons. The most efficient six strains were identified using sequence analysis of the variable D1/D2 domain of the large subunit 26S ribosomal DNA. The identity of these strains was confirmed as Yamadazyma mexicana KKUY-0160, Rhodotorula taiwanensis KKUY-0162, Pichia kluyveri KKUY-0163, Rhodotorula ingeniosa KKUY-0170, Candida pseudointermedia KKUY-0192 and Meyerozyma guilliermondii KKUY-0214. These species are approved for their ability to degrade both aliphatic and aromatic hydrocarbons for the first time in this study. Although, all of them were able to utilize and grow on both hydrocarbons, Rhodotorula taiwanensis KKUY-0162 emerged as the best degrader of octane, and Rhodotorula ingeniosa KKUY-170 was the best degrader of pyrene. GC-MS analysis approved the presence of many chemical compounds that could be transitional or secondary metabolites during the utilization of the hydrocarbons. Our results recommend the application of these yeast species on large scale to approve their efficiency in bioremediation of oil-contamination of the environment. Using these yeasts, either individually or in consortia, could offer a practical solution for aquatic or soil contamination with the crude oil and its derivatives in situ. Copyright © 2017 Elsevier Inc. All rights reserved.

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients

PubMed Central

Garçon, Loïc; Ge, Jingping; Manjunath, Shwetha H.; Mills, Jason A.; Apicella, Marisa; Parikh, Shefali; Sullivan, Lisa M.; Podsakoff, Gregory M.; Gadue, Paul; French, Deborah L.; Mason, Philip J.; Bessler, Monica

2013-01-01

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the “safe harbor” AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells. PMID:23744582

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients.

PubMed

Garçon, Loïc; Ge, Jingping; Manjunath, Shwetha H; Mills, Jason A; Apicella, Marisa; Parikh, Shefali; Sullivan, Lisa M; Podsakoff, Gregory M; Gadue, Paul; French, Deborah L; Mason, Philip J; Bessler, Monica; Weiss, Mitchell J

2013-08-08

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.

DMR1 (CCM1/YGR150C) of Saccharomyces cerevisiae encodes an RNA-binding protein from the pentatricopeptide repeat family required for the maintenance of the mitochondrial 15S ribosomal RNA.

PubMed

Puchta, Olga; Lubas, Michal; Lipinski, Kamil A; Piatkowski, Jakub; Malecki, Michal; Golik, Pawel

2010-04-01

Pentatricopeptide repeat (PPR) proteins form the largest known RNA-binding protein family and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly in mitochondria and chloroplasts, where they modulate organellar genome expression on the post-transcriptional level. The Saccharomyces cerevisiae DMR1 (CCM1, YGR150C) encodes a PPR protein that localizes to mitochondria. Deletion of DMR1 results in a complete and irreversible loss of respiratory capacity and loss of wild-type mtDNA by conversion to rho(-)/rho(0) petites, regardless of the presence of introns in mtDNA. The phenotype of the dmr1Delta mitochondria is characterized by fragmentation of the small subunit mitochondrial rRNA (15S rRNA), that can be reversed by wild-type Dmr1p. Other mitochondrial transcripts, including the large subunit mitochondrial rRNA (21S rRNA), are not affected by the lack of Dmr1p. The purified Dmr1 protein specifically binds to different regions of 15S rRNA in vitro, consistent with the deletion phenotype. Dmr1p is therefore the first yeast PPR protein, which has an rRNA target and is probably involved in the biogenesis of mitochondrial ribosomes and translation.

SCAR Mediates Light-Induced Root Elongation in Arabidopsis through Photoreceptors and Proteasomes[W][OA

PubMed Central

Dyachok, Julia; Zhu, Ling; Liao, Fuqi; He, Ji; Huq, Enamul; Blancaflor, Elison B.

2011-01-01

The ARP2/3 complex, a highly conserved nucleator of F-actin, and its activator, the SCAR complex, are essential for growth in plants and animals. In this article, we present a pathway through which roots of Arabidopsis thaliana directly perceive light to promote their elongation. The ARP2/3-SCAR complex and the maintenance of longitudinally aligned F-actin arrays are crucial components of this pathway. The involvement of the ARP2/3-SCAR complex in light-regulated root growth is supported by our finding that mutants of the SCAR complex subunit BRK1/HSPC300, or other individual subunits of the ARP2/3-SCAR complex, showed a dramatic inhibition of root elongation in the light, which mirrored reduced growth of wild-type roots in the dark. SCAR1 degradation in dark-grown wild-type roots by constitutive photomorphogenic 1 (COP1) E3 ligase and 26S proteasome accompanied the loss of longitudinal F-actin and reduced root growth. Light perceived by the root photoreceptors, cryptochrome and phytochrome, suppressed COP1-mediated SCAR1 degradation. Taken together, our data provide a biochemical explanation for light-induced promotion of root elongation by the ARP2/3-SCAR complex. PMID:21972261

Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

PubMed Central

Kautz, Armin R.; Weisshart, Klaus; Schneider, Annerose; Grosse, Frank; Nasheuer, Heinz-Peter

2001-01-01

Although p48 is the most conserved subunit of mammalian DNA polymerase α-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag. PMID:11507202

Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli.

PubMed Central

Bae, Y M; Holmgren, E; Crawford, I P

1989-01-01

We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzyme of the tryptophan pathway. Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons. This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment. We designate the fused gene trpE(G). The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control. The location of the promoter was determined by S1 nuclease protection, using Rhizobium mRNA. Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained. One of these was a C----T change at position -9 in the -10 region. The +1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site. Images PMID:2656657

Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

PubMed Central

Casutt, Marco S.; Nedielkov, Ruslan; Wendelspiess, Severin; Vossler, Sara; Gerken, Uwe; Murai, Masatoshi; Miyoshi, Hideto; Möller, Heiko M.; Steuber, Julia

2011-01-01

Na+ is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) as the first complex in its respiratory chain. The Na+-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na+ translocation by the Na+-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na+-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na+-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA. PMID:21885438

Two subunits of the 55 K porcine zona pellucida glycoprotein family are immunologically distinct

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

Subramanian, M.G.; Yurewicz, E.C.; Sacco, A.G.

1986-03-01

The 55K glycoprotein family (ZP3) of the porcine zona pellucida is comprised of two subunits of 46 K and 45 K which can be resolved by endo-..beta..-galactosidase digestion of ZP3 followed by reversed phase HPLC on Vydac C4 resin. Gel electrophoresis revealed that the 46 K component (EBDG..cap alpha..) is approx. 95% pure and the 45 K component (EBGD..beta..) is 100% pure. In the present study, these two subunits were evaluated immunologically by RIA. Under similar reaction protocols (chloramine-T iodination procedure) comparable specific activities were obtained for EBGD..cap alpha.. (33.06 +/- 7.5 ..mu..ci/..mu..gm), EBGD..beta.. (30.45 +/- 1.6) and ZP3 (26.3more » +/- 1.3). Antibody (Ab) titration studies revealed that EBGD..cap alpha.. and ..beta.. are potent immunogens and /sup 125/I-EBGD..cap alpha.. showed minimal cross reactivity to EBGD..beta..-Ab (8% bound at 1:500 dilution), whereas, /sup 125/I-EBGD..beta.. showed a greater degree of cross reactivity to EBGD..cap alpha..-Ab (23% bound at 1:500 dilution). Maximum binding for the two labeled antigens against homologous Abs (1:500) was > 60%. Dose response studies revealed that in the /sup 125/I-EBGD..cap alpha.. vs EBGD..cap alpha.. -Ab system, the 50% intercept was 3.25 +/- 0.32 ng for EBGD..cap alpha.. and 472.43 +/- 30.26 ng for EBGD..beta.. (p < 0.01), whereas, in the /sup 125/I-EBGD..beta.. vs EBGD..beta..-Ab system the 50% intercept was 3.51 +/- 0.58 for EBGD..beta.. and 166.77 +/- 49.20 for EBGD..cap alpha.. (p < 0.01). No significant differences were observed in the slopes of the dose response curves. It is concluded that the two subunits of ZP3 possess distinct immunologic characteristics as evaluated by RIA.« less

Protein Flexibility Facilitates Quaternary Structure Assembly and Evolution

PubMed Central

Marsh, Joseph A.; Teichmann, Sarah A.

2014-01-01

The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components. PMID:24866000

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

PubMed

Mundus, D; Wollenzien, P

1998-11-01

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex* ♦

PubMed Central

Nadeau, Owen W.; Lane, Laura A.; Xu, Dong; Sage, Jessica; Priddy, Timothy S.; Artigues, Antonio; Villar, Maria T.; Yang, Qing; Robinson, Carol V.; Zhang, Yang; Carlson, Gerald M.

2012-01-01

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)4 complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)2 lobes joined with D2 symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)4 complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)4 kinase core, because a β4 subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit. PMID:22969083

Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

PubMed

Mellor, J R; Wisden, W; Randall, A D

2000-07-10

Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden, W., 1997. Ligand-gated ion channel partnerships: GABA(A) receptor alpha(6) subunit inactivation inhibits delta subunit expression. Journal of Neuroscience 17, 1350-1362).

YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

PubMed Central

Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

2014-01-01

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

Receptor model for the molecular basis of tissue selectivity of 1,4-dihydropyridine calcium channel drugs

NASA Astrophysics Data System (ADS)

Langs, David A.; Strong, Phyllis D.; Triggle, David J.

1990-09-01

Our analysis of the solid state conformations of nifedipine [dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinecarboxylate] and its 1,4-dihydropyridine (1,4-DHP) analogues produced a cartoon description of the important interactions between these drugs and their voltage-dependent calcium channel receptor. In the present study a molecular-level detailed model of the 1,4-DHP receptor binding site has been built from the published amino acid sequence of the 215-1 subunit of the voltage-dependent calcium channel isolated from rabbit skeletal muscle transverse tubule membranes. The voltage-sensing component of the channel described in this work differs from others reported for the homologous sodium channel in that it incorporates a water structure and a staggered, rather than eclipsed, hydrogen bonded S4 helix conformation. The major recognition surfaces of the receptor lie in helical grooves on the S4 or voltagesensing α-helix that is positioned in the center of the bundle of transmembrane helices that define each of the four calcium channel domains. Multiple binding clefts defined by Arg-X-X-Arg-P-X-X-S `reading frames' exist on the S4 strand. The tissue selectivity of nifedipine and its analogues may arise, in part, from conservative changes in the amino acid residues at the P and S positions of the reading frame that define the ester-binding regions of receptors from different tissues. The crystal structures of two tissue-selective nifedipine analogues, nimodipine [isopropyl (2-methoxyethyl) 1,4-dihydro-2,6- dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylate] and nitrendipine [ethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylate] are reported. Nimodipine was observed to have an unusual ester side chain conformation that enhances the fit to the proposed ester-sensing region of the receptor.

The Barley Magnesium Chelatase 150-kD Subunit Is Not an Abscisic Acid Receptor1[OA

PubMed Central

Müller, André H.; Hansson, Mats

2009-01-01

Magnesium chelatase is the first unique enzyme of the chlorophyll biosynthetic pathway. It is composed of three gene products of which the largest is 150 kD. This protein was recently identified as an abscisic acid receptor in Arabidopsis (Arabidopsis thaliana). We have evaluated whether the barley (Hordeum vulgare) magnesium chelatase large subunit, XanF, could be a receptor for the phytohormone. The study involved analysis of recombinant magnesium chelatase protein as well as several induced chlorophyll-deficient magnesium chelatase mutants with defects identified at the gene and protein levels. Abscisic acid had no effect on magnesium chelatase activity and binding to the barley 150-kD protein could not be shown. Magnesium chelatase mutants showed a wild-type response in respect to postgermination growth and stomatal aperture. Our results question the function of the large magnesium chelatase subunit as an abscisic acid receptor. PMID:19176716

Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

PubMed Central

Shen, Peter S.; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H.; Cox, James; Cheng, Yifan; Lambowitz, Alan M.; Weissman, Jonathan S.; Brandman, Onn; Frost, Adam

2015-01-01

In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”). PMID:25554787

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1

NASA Technical Reports Server (NTRS)

Henry, R. L.; Takemoto, L. J.; Murphy, J.; Gallegos, G. L.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.

Differentially expressed proteins in nitric oxide-stimulated NIH/3T3 fibroblasts: implications for inhibiting cancer development.

PubMed

Shim, Dong Hwi; Lim, Joo Weon; Kim, Hyeyoung

2015-03-01

Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. The cells were treated with 300 μM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (α1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (ε subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (ε subunit) are unknown in relation to carcinogenesis. Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

Functional conservation of RNA polymerase II in fission and budding yeasts.

PubMed

Shpakovski, G V; Gadal, O; Labarre-Mariotte, S; Lebedenko, E N; Miklos, I; Sakurai, H; Proshkin, S A; Van Mullem, V; Ishihama, A; Thuriaux, P

2000-02-04

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits. Copyright 2000 Academic Press.

Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

ERIC Educational Resources Information Center

Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

2016-01-01

Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

Platelet antiheparin activity. The isolation and characterisation of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity.

PubMed

Moore, S; Pepper, D S; Cash, J D

1975-02-27

Platelet factor 4 was isolated by gel filtration from the soluble release products of thrombin-aggregated washed human platelets as a proteoglycan-platelet factor 4 complex of molecular weight 358 000, Stokes radius (r-s) of 14.0 nm, sedimentation coefficient (s) of 7.1 S and frictional ratio (f/f-o) of 3.04. The complex was dissociated at high ionic strength (I equals 0.75) and the proteoglycan separated from platelet factor 4 by gel filtration. Platelet factor 4 had a molecular weight of 27 100, r-s of 2.52 nm, s of 2.4 S and f/f-o of 1.26, was insoluble under physiological conditions but readily soluble at pH 3. Under these conditions platelet factor 4 dissociated into four subunits with a molecular weight of 6900, r-s of 1.92 nm, s of 0.8 S, and f/f-o of 1.52. Qualitative N-terminal amino acid analysis showed the presence of glutamic acid or glutamine as the major end group. Platelet factor 4 was compared with protamine sulphate, which has similar biological properties, by electrophoresis at pH 2.2, in which both migrated as single bands but with differing mobility, and by amino acid analysis which showed a more normal distribution of residues than occurred in protamine sulphate. Of the basic amino acids platelet factor 4 (molecular weight 27 100) contained 5.97% arginine, 3.18% histidine, and 12.31% lysine compared to protamine sulphate with 64.2% arginine, 0.6% lysine and no histidine. A partial specific volume (v) of 0.747 was calculated for platelet factor 4 from its amino acid analysis. A membrane fraction with antiheparin activity, an isopycnic density of 1.090-1.110 and r-s of 15-35 nm, was also isolated by sucrose density gradient centrifugation from the ultrasonicated insoluble platelet residue remaining after thrombin-induced aggregation of washed human platelets. Trypsin treatment of the membrane fraction neither solubilised nor destroyed the activity.

beta subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line.

PubMed

Moreno, H; Rudy, B; Llinás, R

1997-12-09

Human epithelial kidney cells (HEK) were prepared to coexpress alpha1A, alpha2delta with different beta calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney alpha1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of alpha1A, betaIb, and alpha2delta produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of omega-agatoxin IVA (omega-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by alpha1A, beta2a, alpha2delta subunits, which demonstrated the slowest inactivation and were relatively insensitive to omega-Aga IVA and sFTX. Coexpression of beta3 with the same combination as above produced inactivating currents also insensitive to low concentration of omega-Aga IVA and sFTX. These data indicate that the combination alpha1A, betaIb, alpha2delta best resembles P-type channels given the rate of inactivation and the high sensitivity to omega-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the beta subunit associated with the alpha1A subunit.

Molecular Characterization and Functional Analysis of a Ferritin Heavy Chain Subunit from the Eri-Silkworm, Samia cynthia ricini

PubMed Central

Yu, Hai-Zhong; Zhang, Shang-Zhi; Ma, Yan; Fei, Dong-Qiong; Li, Bing; Yang, Li-Ang; Wang, Jie; Li, Zhen; Muhammad, Azharuddin; Xu, Jia-Ping

2017-01-01

Ferritins are conserved iron-binding proteins that are primarily involved in iron storage, detoxification and the immune response. Despite the importance of ferritin in organisms, little is known about their roles in the eri-silkworm (Samia cynthia ricini). We previously identified a ferritin heavy chain subunit named ScFerHCH in the S. c. ricini transcriptome database. The full-length S. c. ricini ferritin heavy chain subunit (ScFerHCH) was 1863 bp and encoded a protein of 231 amino acids with a deduced molecular weight of 25.89 kDa. Phylogenetic analysis revealed that ScFerHCH shared a high amino acid identity with the Bombyx mori and Danaus plexippus heavy chain subunits. Higher ScFerHCH expression levels were found in the silk gland, fat body and midgut of S. c. ricini by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Injection of Staphylococcus aureus and Pseudomonas aeruginosa was associated with an upregulation of ScFerHCH in the midgut, fat body and hemolymph, indicating that ScFerHCH may contribute to the host’s defense against invading pathogens. In addition, the anti-oxidation activity and iron-binding capacity of recombinant ScFerHCH protein were examined. Taken together, our results suggest that the ferritin heavy chain subunit from eri-silkworm may play critical roles not only in innate immune defense, but also in organismic iron homeostasis. PMID:29036914

Alcohol modulation of BK channel gating depends on β subunit composition

PubMed Central

Kuntamallappanavar, Guruprasad

2016-01-01

In most mammalian tissues, Ca2+i/voltage-gated, large conductance K+ (BK) channels consist of channel-forming slo1 and auxiliary (β1–β4) subunits. When Ca2+i (3–20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca2+i concentrations—all determinants of the BK response to ethanol—made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel (“cbv1” from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca2+i while inhibiting current at high Ca2+i, the potentiation–inhibition crossover occurring at 20 µM Ca2+i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca2+i. Second, applying Horrigan–Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel’s apparent Ca2+ affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca2+ binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd, yet this decrease (two times) is much smaller than that of cbv1; (b) reduces C; and (c) decreases coupling between Ca2+ binding and voltage sensing (parameter E). Decreased allosteric coupling leads to diminished BK activity. Thus, we have identified critical gating modifications that lead to the differential actions of ethanol on slo1 with and without different β subunits. PMID:27799321

Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

PubMed

Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

2008-11-01

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

Triazine herbicide resistance in the photosynthetic bacterium Rhodopseudomonas sphaeroides

PubMed Central

Brown, Alfred E.; Gilbert, Carl W.; Guy, Rachel; Arntzen, Charles J.

1984-01-01

The photoaffinity herbicide azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine) selectively labels the L subunit of the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides. Herbicide-resistant mutants retain the L subunit and have altered binding properties for methylthio- and chloro-substituted triazines as well as altered equilibrium constants for electron transfer between primary and secondary electron acceptors. We suggest that a subtle alteration in the L subunit is responsible for herbicide resistance and that the L subunit is the functional analog of the 32-kDa QB protein of chloroplast membranes. Images PMID:16593520