Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230*

PubMed Central

MacDonald, Nicholas J.; Nguyen, Vu; Shimp, Richard; Reiter, Karine; Herrera, Raul; Burkhardt, Martin; Muratova, Olga; Kumar, Krishan; Aebig, Joan; Rausch, Kelly; Lambert, Lynn; Dawson, Nikiah; Sattabongkot, Jetsumon; Ambroggio, Xavier; Duffy, Patrick E.; Wu, Yimin; Narum, David L.

2016-01-01

Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria. PMID:27432885

Introgression of leginsulin, a cysteine-rich protein, and high-protein trait from an Asian soybean plant introduction genotype into a North American experimental soybean line

USDA-ARS?s Scientific Manuscript database

Soybean is an important protein source for both humans and animals. However, soybean proteins are relatively poor in the sulfur-containing amino acids, cysteine and methionine. Improving the content of endogenous proteins rich in sulfur containing amino acids could enhance the nutritive value of soy...

A New Family of Giardial Cysteine-Rich Non-VSP Protein Genes and a Novel Cyst Protein

PubMed Central

Birkeland, Shanda R.; Preheim, Sarah P.; Cipriano, Michael J.; McArthur, Andrew G.; Gillin, Frances D.

2006-01-01

Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, “HCNCp” (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many “CxxC” tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple “CxC” motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp C-terminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing ≥20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

PubMed Central

Hansen, M S; Barklis, E

1995-01-01

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC. PMID:7815493

Purification, crystallization and preliminary X-ray crystallographic analysis of a cysteine-rich secretory protein (CRISP) from Naja atra venom.

PubMed

Wang, Yu-Ling; Goh, King-Xiang; Wu, Wen-guey; Chen, Chun-Jung

2004-10-01

Cysteine-rich secretory proteins (CRISPs) play an important role in the innate immune system and are transcriptionally regulated by androgens in several tissues. The proteins are mostly found in the epididymis and granules of mammals, whilst a number of snake venoms also contain CRISP-family proteins. The natrin protein from the venom of Naja atra (Taiwan cobra), which belongs to a family of CRISPs and has a cysteine-rich C-terminal amino-acid sequence, has been purified using a three-stage chromatography procedure and crystals suitable for X-ray analysis have been obtained using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.58 A resolution using synchrotron radiation; the crystals belong to space group C222(1), with unit-cell parameters a = 59.172, b = 65.038, c = 243.156 A. There are two protein molecules in the asymmetric unit and the Matthews coefficient is estimated to be 2.35 A3 Da(-1), corresponding to a solvent content of 47.60%.

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins*

PubMed Central

Meitzler, Jennifer L.; Hinde, Sara; Bánfi, Botond; Nauseef, William M.; Ortiz de Montellano, Paul R.

2013-01-01

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1. PMID:23362256

Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain

DOE PAGES

Travers, Timothy; Lopez Bautista, Cesar Augusto; Van, Que; ...

2018-05-31

Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here in this paper, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which ismore » consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.« less

Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain

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

Travers, Timothy; Lopez Bautista, Cesar Augusto; Van, Que

Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here in this paper, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which ismore » consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.« less

Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly*

PubMed Central

Smulski, Cristian R.; Beyrath, Julien; Decossas, Marion; Chekkat, Neila; Wolff, Philippe; Estieu-Gionnet, Karine; Guichard, Gilles; Speiser, Daniel; Schneider, Pascal; Fournel, Sylvie

2013-01-01

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a KD of 0.6 μm. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state. PMID:23463508

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli

PubMed Central

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2014-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodesricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of 1H–15N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in 13C/15N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins. PMID:25628987

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

PubMed

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2015-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

An ELISA for SGP28/CRISP-3, a cysteine-rich secretory protein in human neutrophils, plasma, and exocrine secretions.

PubMed

Udby, Lene; Cowland, Jack B; Johnsen, Anders H; Sørensen, Ole E; Borregaard, Niels; Kjeldsen, Lars

2002-05-01

Specific granule protein of 28 kDa (SGP28), also termed cysteine-rich secretory protein 3 (CRISP-3), is a glycoprotein that belongs to a family of cysteine-rich secretory proteins (CRISPs). SGP28 was originally discovered in human neutrophils, but transcripts are widely distributed in exocrine glands (salivary glands, pancreas, and prostate) and also found at lower levels in epididymis, ovary, thymus, and colon. The function of SGP28/CRISP-3 is not yet known. Similarities to pathogenesis-related proteins in plants and the expression in neutrophils and exocrine glands suggest that SGP28/CRISP-3 may play a role in innate host defense. We describe here the production of a recombinant, C-terminally truncated form of CRISP-3 (rCRISP-3Delta) and the generation of polyclonal antibodies against rCRISP-3Delta that are useful in immunoblotting and immunocytochemistry. We present a specific, accurate, and reproducible enzyme-linked immunosorbant assay (ELISA) for the measurement of CRISP-3 with a detection limit of 2 ng/ml. We further demonstrate the presence of CRISP-3 protein in human plasma (6.3 microg/ml), saliva (21.8 microg/ml), seminal plasma (11.2 microg/ml), and sweat (0.15 microg/ml), and describe the coexistence of two different molecular weight forms of CRISP-3, representing an N-glycosylated and a non-glycosylated form of the mature protein.

The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

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

Bradshaw, William J.; Public Health England, Porton Down, Salisbury SP4 0JG; Kirby, Jonathan M.

2014-07-01

The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA)more » into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.« less

Cysteine-rich secretory proteins (CRISP) and their role in mammalian fertilization.

PubMed

Cohen, Débora J; Maldera, Julieta A; Weigel Muñoz, Mariana; Ernesto, Juan I; Vasen, Gustavo; Cuasnicu, Patricia S

2011-01-01

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.

Genome-wide search and comparative genomic analysis of the trypsin inhibitor-like cysteine-rich domain-containing peptides.

PubMed

Zeng, Xian-Chun; Liu, Yichen; Shi, Wanxia; Zhang, Lei; Luo, Xuesong; Nie, Yao; Yang, Ye

2014-03-01

It was shown that peptides containing trypsin inhibitor-like cysteine-rich (TIL) domain are able to inhibit proteinase activities, and thus play important roles in various biological processes, such as immune response and anticoagulation. However, only a limited number of the TIL peptides have been identified and characterized so far; and little has been known about the evolutionary relationships of the genes encoding the TIL peptides. BmKAPi is a TIL domain-containing peptide that was identified from Mesobuthus martensii Karsch. Here, we conducted genome-wide searches for new peptides that are homologous to BmKAPi or possess a cysteine pattern similar to that of BmKAPi. As a result, we identified a total of 80 different TIL peptides from 34 species of arthropods. We found that these peptides can be classified into seven evolutionarily distinct groups. Furthermore, we cloned the genomic sequence of BmKAPi; the genomic sequences of the majority of other TIL peptides were also identified from the GenBank database using bioinformatical approaches. Through phylogenetic and comparative genomic analysis, we found 26 cases of intron gain events occurred in the genes of the TIL peptides; however, no instances of intron loss were observed. Moreover, we found that alternative splicing contributes to the diversification of the TIL peptides. It is interesting to see that four genes of the TIL domain-containing peptides overlap in a DNA region located on the chromosome LG B15 of Bombus terretris. These data suggest that the evolution of the TIL peptide genes are dynamic, which was dominated by intron gain. Copyright © 2013 Elsevier Inc. All rights reserved.

Cysteine-rich buccal gland protein suppressed the proliferation, migration and invasion of hela cells through akt pathway.

PubMed

Han, Jianmei; Liu, Yu; Jiang, Qi; Xiao, Rong

2017-11-01

Cysteine-rich buccal gland protein (CRBGP) as a member of cysteine-rich secretory proteins (CRISPs) superfamily was isolated from the buccal glands of Lampetra japonica, the blood suckers in the marine. Previous studies showed CRBGP could suppress angiogenesis probably due to its ion channel blocking activity. Whether CRBGP could also affect the activity of tumor cells has not been reported yet. In this study, CRBGP suppressed the proliferation of Hela cells with an IC 50 of 6.7 μM by inducing apoptosis. Both microscopic observation and Western blot indicated that CRBGP was able to induce the nuclei shrinking, downregulate the protein level of BCL2 and caspase 3 as well as upregulate the level of BAX in Hela cells, suggested that CRBGP might induce apoptosis of Hela cells in a mitochondrial-dependent pathway. Furthermore, CRBGP could disturb F-actin organization, which would finally cause the Hela cells to lose their shape and to lessen their abilities on adhesion, migration and invasion. Finally, CRBGP was shown to reduce the phosphorylation level of Akt, which indicated that CRBGP might inhibit the proliferation and metastasis of Hela cells through Akt pathway. CRBGP, as a voltage-gated sodium channel blocker, also possesses the anti-tumor abilities which provided information on the effects and action manner of the other CRISPs. © 2017 IUBMB Life, 69(11):856-866, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Solution structure of the catalytic domain of RICH protein from goldfish.

PubMed

Kozlov, Guennadi; Denisov, Alexey Y; Pomerantseva, Ekaterina; Gravel, Michel; Braun, Peter E; Gehring, Kalle

2007-03-01

Regeneration-induced CNPase homolog (RICH) is an axonal growth-associated protein, which is induced in teleost fish upon optical nerve injury. RICH consists of a highly acidic N-terminal domain, a catalytic domain with 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity and a C-terminal isoprenylation site. In vitro RICH and mammalian brain CNPase specifically catalyze the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains unknown. Here, we report the NMR structure of the catalytic domain of goldfish RICH and describe its binding to CNPase inhibitors. The structure consists of a twisted nine-stranded antiparallel beta-sheet surrounded by alpha-helices on both sides. Despite significant local differences mostly arising from a seven-residue insert in the RICH sequence, the active site region is highly similar to that of human CNPase. Likewise, refinement of the catalytic domain of rat CNPase using residual dipolar couplings gave improved agreement with the published crystal structure. NMR titrations of RICH with inhibitors point to a similar catalytic mechanism for RICH and CNPase. The results suggest a functional importance for the evolutionarily conserved phosphodiesterase activity and hint of a link with pre-tRNA splicing.

Cysteine-Rich Atrial Secretory Protein from the Snail Achatina achatina: Purification and Structural Characterization

PubMed Central

Shabelnikov, Sergey; Kiselev, Artem

2015-01-01

Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported. In this study, we describe an 18.1-kDa, cysteine-rich atrial secretory protein (CRASP) isolated from the terrestrial snail Achatina achatina that has no detectable sequence similarity to any known protein or nucleotide sequence. CRASP is an acidic, 158-residue, N-glycosylated protein composed of eight alpha-helical segments stabilized with five disulphide bonds. A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold. CRASP is most strongly expressed in the atrium in secretory atrial granular cells, and substantial amounts of CRASP are released from the heart upon nerve stimulation. CRASP is detected in the haemolymph of intact animals at nanomolar concentrations. CRASP is the first secretory protein expressed in molluscan atrium to be reported. We propose that CRASP is an example of a taxonomically restricted gene that might be responsible for adaptations specific for terrestrial pulmonates. PMID:26444993

Small cysteine-rich antifungal proteins from radish: their role in host defense.

PubMed Central

Terras, F R; Eggermont, K; Kovaleva, V; Raikhel, N V; Osborn, R W; Kester, A; Rees, S B; Torrekens, S; Van Leuven, F; Vanderleyden, J

1995-01-01

Radish seeds have previously been shown to contain two homologous, 5-kD cysteine-rich proteins designated Raphanus sativus-antifungal protein 1 (Rs-AFP1) and Rs-AFP2, both of which exhibit potent antifungal activity in vitro. We now demonstrate that these proteins are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs. Moreover, Rs-AFPs are preferentially released during seed germination after disruption of the seed coat. The amount of released proteins is sufficient to create a microenvironment around the seed in which fungal growth is suppressed. Both the cDNAs and the intron-containing genomic regions encoding the Rs-AFP preproteins were cloned. Transcripts (0.55 kb) hybridizing with an Rs-AFP1 cDNA-derived probe were present in near-mature and mature seeds. Such transcripts as well as the corresponding proteins were barely detectable in healthy uninfected leaves but accumulated systemically at high levels after localized fungal infection. The induced leaf proteins (designated Rs-AFP3 and Rs-AFP4) were purified and shown to be homologous to seed Rs-AFPs and to exert similar antifungal activity in vitro. A chimeric Rs-AFP2 gene under the control of the constitutive cauliflower mosaic virus 35S promoter conferred enhanced resistance to the foliar pathogen Alternaria longipes in transgenic tobacco. The term "plant defensins" is proposed to denote these defense-related proteins. PMID:7780308

Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins.

PubMed

Valdivieso-Torres, Leonardo; Sarangi, Anindita; Whidby, Jillian; Marcotrigiano, Joseph; Roth, Monica J

2015-12-30

Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines

Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis

NASA Astrophysics Data System (ADS)

Yang, Kun; Rong, Wei; Qi, Lin; Li, Jiarui; Wei, Xuening; Zhang, Zengyan

2013-10-01

Cysteine-rich receptor kinases (CRKs) belong to the receptor-like kinase family. Little is known about CRK genes in wheat. We isolated a wheat CRK gene TaCRK1 from Rhizoctonia cerealis-resistant wheat CI12633 based on a differentially expressed sequence identified by RNA-Sequencing (RNA-Seq) analysis. TaCRK1 was more highly expressed in CI12633 than in susceptible Wenmai 6. Transcription of TaCRK1 in wheat was induced in CI12633 after R. cerealis infection and exogenous abscisic acid (ABA) treatment. The deduced TaCRK1 protein contained a signal peptide, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase domain. Transient expression of a green fluorescence protein fused with TaCRK1 in wheat and onion indicated that TaCRK1 may localize to plasma membranes. Characterization of TaCRK1 silencing induced by virus-mediated method in CI12633 showed that the downregulation of TaCRK1 transcript did not obviously impair resistance to R. cerealis. This study paves the way to further CRK research in wheat.

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

PubMed Central

Lidell, Martin E.; Moncada, Darcy M.; Chadee, Kris; Hansson, Gunnar C.

2006-01-01

In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel. PMID:16754877

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel.

PubMed

Lidell, Martin E; Moncada, Darcy M; Chadee, Kris; Hansson, Gunnar C

2006-06-13

In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.

Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation.

PubMed

Chen, P; Melchior, C; Brons, N H; Schlegel, N; Caen, J; Kieffer, N

2001-10-19

We have investigated receptor function and epitope expression of recombinant alpha(IIb)beta(3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to alpha(IIb)beta(3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha(IIb)beta(3)C273A, but interacted only weakly with mutant alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin

A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced From Studies With Subunit B of Heterodisulfide Reductase From Methanothermobacter Marburgensis

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

Hamann, N.; Mander, G.J.; Shokes, J.E.

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]{sup 3+} cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX{sub 31-39}CCX{sub 35-36}CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusualmore » [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (g{sub zyx} = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. {sup 57}Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with {sup 57}Fe hyperfine couplings very similar to that of CoM-HDR. CoM-{sup 33}SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S{sub 3}(O/N){sub 1} geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to

Participation of cysteine-rich secretory proteins (CRISP) in mammalian sperm-egg interaction.

PubMed

Cohen, Débora J; Busso, Dolores; Da Ros, Vanina; Ellerman, Diego A; Maldera, Julieta A; Goldweic, Nadia; Cuasnicu, Patricia S

2008-01-01

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. CRISP1 (cysteine-rich secretory protein 1) is an epididymal protein thought to participate in gamete fusion through its binding to egg-complementary sites. Structure-function studies using recombinant fragments of CRISP1 as well as synthetic peptides reveal that its egg-binding ability resides in a 12 amino acid region corresponding to an evolutionary conserved motif of the CRISP family, named Signature 2 (S2). Further experiments analyzing both the ability of other CRISP proteins to bind to the rat egg and the amino acid sequence of their S2 regions show that the amino acid sequence of the S2 is needed for CRISP1 to interact with the egg. CRISP1 appears to be involved in the first step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. The observation that sperm testicular CRISP2 is also able to bind to the egg surface suggests a role for this protein in gamete fusion. Subsequent experiments confirmed the participation of CRISP2 in this step of fertilization and revealed that CRISP1 and CRISP2 interact with common egg surface binding sites. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization. These observations contribute to a better understanding of the molecular mechanisms underlying mammalian fertilization.

Protein Topology Determines Cysteine Oxidation Fate: The Case of Sulfenyl Amide Formation among Protein Families

PubMed Central

Defelipe, Lucas A.; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A.; Turjanski, Adrián G.

2015-01-01

Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692

Characterization of a family of cysteine rich proteins and development of a MaSp1 derived miniature fibroin

NASA Astrophysics Data System (ADS)

Chuang, Tyler Casey

Spider silk displays a unique balance of high tensile strength and extensibility, making it one of the toughest materials on the planet. Dragline silk, also known as the lifeline of the spider, represents one of the best studied fiber types and many labs are attempting to produce synthetic dragline silk fibers for commercial applications. In these studies, we develop a minifibroin for expression studies in bacteria. Using recombinant DNA methodology and protein expression studies, we develop a natural minifibroin that contains the highly conserved N- and C-terminal domains, along with several internal block repeats of MaSp1. We also characterize a family of small cysteine-rich proteins (CRPs) and demonstrate that these factors are present within the spinning dope of the major ampullate gland using MS analysis. Biochemical studies and characterization of one of the family members, CRP1, demonstrate that this factor can self-polymerize into higher molecular weight complexes under oxidizing conditions, but can be converted into a monomeric species under reducing conditions. Self-polymerization of CRP1 is also shown to be independent of pH and salt concentration, two important chemical cues that help fibroin aggregation. Overall, our data demonstrate that the polymerization state of CRP1 is dependent upon redox state, suggesting that the redox environment during fiber extrusion may help regulate the oligomerization of CRP molecules during dragline silk production.

The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

PubMed

Wirtz, M; Berkowitz, O; Droux, M; Hell, R

2001-02-01

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.

Structures of class A macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich domain.

PubMed

Resnick, D; Chatterton, J E; Schwartz, K; Slayter, H; Krieger, M

1996-10-25

Structures of secreted forms of the human type I and II class A macrophage scavenger receptors were studied using biochemical and biophysical methods. Proteolytic analysis was used to determine the intramolecular disulfide bonds in the type I-specific scavenger receptor cysteine-rich (SRCR) domain: Cys2-Cys7, Cys3-Cys8, and Cys5-Cys6. This pattern is likely to be shared by the highly homologous domains in the many other members of the SRCR domain superfamily. Electron microscopy using rotary shadowing and negative staining showed that the type I and II receptors are extended molecules whose contour lengths are approximately 440 A. They comprised two adjacent fibrous segments, an alpha-helical coiled-coil ( approximately 230 A, including a contribution from the N-terminal spacer domain) and a collagenous triple helix ( approximately 210 A). The type I molecules also contained a C-terminal globular structure ( approximately 58 x 76 A) composed of three SRCR domains. The fibrous domains were joined by an extremely flexible hinge. The angle between these domains varied from 0 to 180 degrees and depended on the conditions of sample preparation. Unexpectedly, at physiologic pH, the prevalent angle seen using rotary shadowing was 0 degrees , resulting in a structure that is significantly more compact than previously suggested. The apparent juxtaposition of the fibrous domains at neutral pH provides a framework for future structure-function studies of these unusual multiligand receptors.

Bleogens: Cactus-Derived Anti-Candida Cysteine-Rich Peptides with Three Different Precursor Arrangements

PubMed Central

Loo, Shining; Kam, Antony; Xiao, Tianshu; Tam, James P.

2017-01-01

Cysteine-rich peptides (CRPs) play important host-defense roles in plants. However, information concerning CRPs in the Cactaceae (cactus) family is limited, with only a single cactus-derived CRP described to date. Here, we report the identification of 15 novel CRPs with three different precursor architectures, bleogens pB1-15 from Pereskia bleo of the Cactaceae family. By combining proteomic and transcriptomic methods, we showed that the prototype, bleogen pB1, contained 36 amino acid residues, a six-cysteine motif typical of the six-cysteine-hevein-like peptide (6C-HLP) family, and a type I two-domain precursor consisting of an endoplasmic reticulum (ER) and a mature domain. In contrast, the precursors of the other 14 bleogens contained a type II three-domain architecture with a propeptide domain inserted between the ER and the mature bleogen domain. Four of these 14 bleogens display a third type of architecture with a tandemly repeating bleogen domain. A search of the Onekp database revealed that <1% plant species possess three different precursor architectures for the biosynthesis of 6C-HLPs, including Lophophora williamsii, Pereskia aculeate, Portulaca cryptopetala, Portulaca oleracea, Portulaca suffruticosa, and Talinum sp. NMR analysis confirmed that bleogen pB1 has cystine-knot disulfide connectivity as well as a two-beta-sheet and a four-loop structural fold that is similar to other 6C-HLPs. Sequence analysis, structural studies, and in silico modeling revealed that bleogen pB1 has a cation-polar-cation motif, a signature heparin-binding motif that was confirmed by heparin affinity chromatography. Cell-based assays showed that bleogen pB1 is non-toxic to mammalian cells but functions as an anti-Candida peptide. Taken together, our findings provide insight into the occurrence, functions and precursor architectures of CRPs in the cactus family. PMID:29312404

Blends of cysteine-containing proteins

NASA Astrophysics Data System (ADS)

Barone, Justin

2005-03-01

Many agricultural wastes are made of proteins such as keratin, lactalbumin, gluten, and albumin. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter-and intra-molecular sulfur-sulfur bonds. Correlations are made between the properties of films made from the proteins and the amino acid sequence. Blends of cysteine-containing proteins show possible synergies in physical properties at intermediate concentrations. FT-IR spectroscopy shows increased hydrogen bonding at intermediate concentrations suggesting that this contributes to increased physical properties. DSC shows limited miscibility and the formation of new crystalline phases in the blends suggesting that this too contributes.

Secreted Protein Acidic and Rich in Cysteine (SPARC) in Human Skeletal Muscle

PubMed Central

Jørgensen, Louise H.; Petersson, Stine J.; Sellathurai, Jeeva; Andersen, Ditte C.; Thayssen, Susanne; Sant, Dorte J.; Jensen, Charlotte H.; Schrøder, Henrik D.

2009-01-01

Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15–16 weeks. SPARC protein was detected in the majority of analyzed muscle biopsies (23 of 24), mainly in mononuclear cells of which few were pax7 positive. Myotubes and regenerating myofibers also expressed SPARC. The expression-degree seemed to reflect the severity of the lesion. In accordance with these in vivo findings, primary human-derived satellite cells were found to express SPARC both during proliferation and differentiation in vitro. In conclusion, this study shows SPARC expression both during muscle development and in regenerating muscle. The expression is detected both in satellite cells/myoblasts and in myotubes and muscle fibers, indicating a role for SPARC in the skeletal muscle compartment. (J Histochem Cytochem 57:29–39, 2009) PMID:18796407

Emergence of CD134 cysteine-rich domain 2 (CRD2)-independent strains of feline immunodeficiency virus (FIV) is associated with disease progression in naturally infected cats.

PubMed

Bęczkowski, Paweł M; Techakriengkrai, Navapon; Logan, Nicola; McMonagle, Elizabeth; Litster, Annette; Willett, Brian J; Hosie, Margaret J

2014-11-28

Feline immunodeficiency virus (FIV) infection is mediated by sequential interactions with CD134 and CXCR4. Field strains of virus vary in their dependence on cysteine-rich domain 2 (CRD2) of CD134 for infection. Here, we analyse the receptor usage of viral variants in the blood of 39 naturally infected cats, revealing that CRD2-dependent viral variants dominate in early infection, evolving towards CRD2-independence with disease progression. These findings are consistent with a shift in CRD2 of CD134 usage with disease progression.

Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example

PubMed Central

Kim, Yong Joon; Kaluz, Stefan; Mehta, Anil; Weinert, Emily; Rivera, Shannon; Van Meir, Erwin G.

2017-01-01

The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies. p300-CH1 has 3 zinc fingers and 9 cysteine residues, posing challenges associated with reagent compatibility and protein oxidation. A protocol has been developed to overcome such issues by incorporating zinc during expression and streamlining the purification time, resulting in a high yield of optimally folded protein (120 mg per 4 L expression media) that is suitable for structural NMR studies. The structural integrity of the final recombinant p300-CH1 has been verified to be optimal using onedimensional 1H NMR spectroscopy and circular dichroism. This protocol is applicable for the purification of other zinc finger containing proteins. PMID:28966947

[Plant signaling peptides. Cysteine-rich peptides].

PubMed

Ostrowski, Maciej; Kowalczyk, Stanisław

2015-01-01

Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation.

Characterization and localization of cysteine-rich secretory protein 3 (CRISP-3) in the human male reproductive tract.

PubMed

Udby, Lene; Bjartell, Anders; Malm, Johan; Egesten, Arne; Lundwall, Ake; Cowland, Jack B; Borregaard, Niels; Kjeldsen, Lars

2005-01-01

Mammalian members of the cysteine-rich secretory protein (CRISP) family are expressed predominantly in the male reproductive tract and are implicated in the process of reproduction from spermiogenesis, posttesticular sperm maturation, and capacitation to oocyte-sperm fusion, and possibly also penetration of the zona pellucida. Rodents express only 2 CRISPs (CRISP-1 and CRISP-2) in their male reproductive system, whereas humans and horses express an additional third member named CRISP-3. We have previously demonstrated that this protein is present in human seminal plasma as well as in other exocrine secretions, in blood plasma, and in neutrophilic granulocytes. To characterize the protein in seminal plasma and localize the production of CRISP-3 in the human male reproductive tract, we performed immunoblotting and enzyme-linked immunosorbent assay measurements of seminal plasma and immunohistochemistry and in situ hybridization of tissue specimens. We were able to show that human CRISP-3 is a quantitatively minor seminal plasma protein not associated with prostasomes. Furthermore, CRISP-3 expression was found in the secretory epithelium throughout the male genital tract, with particularly high expression in the cauda epididymis and ampulla vas deferens. Examination of seminal plasma from vasectomized males indicates that organs downstream of the epididymis are probably the major sources of seminal plasma CRISP-3.

Mouse cysteine-rich secretory protein 4 (CRISP4): a member of the Crisp family exclusively expressed in the epididymis in an androgen-dependent manner.

PubMed

Jalkanen, Jenni; Huhtaniemi, Ilpo; Poutanen, Matti

2005-05-01

The final maturation of spermatozoa produced in the testis takes place during their passage through the epididymis. In this process, the proteins secreted into the epididymal lumen along with changes in the pH and salt composition of the epididymal fluid cause several biochemical changes and remodeling of the sperm plasma membrane. The Crisp family is a group of cysteine-rich secretory proteins that previously consisted of three members, one of which-CRISP1-is an epididymal protein shown to attach to the sperm surface in the epididymal lumen and to inhibit gamete membrane fusion. In the present paper, we introduce a new member of the Crisp protein family, CRISP4. The new gene was discovered through in silico analysis of the epididymal expressed sequence tag library deposited in the UniGene database. The peptide sequence of CRISP4 has a signal sequence suggesting that it is secreted into the epididymal lumen and might thus interact with sperm. Unlike the other members of the family, Crisp4 is located on chromosome 1 in a cluster of genes encoding for cysteine-rich proteins. Crisp4 is expressed in the mouse exclusively in epithelial cells of the epididymis in an androgen-dependent manner, and the expression of the gene starts at puberty along with the onset of sperm maturation. The identified murine CRISP4 peptide has high homology with human CRISP1, and the homology is higher than that between murine and human CRISP1, suggesting that CRISP4 represents the mouse counterpart of human CRISP1 and could have similar effects on sperm membrane as mouse and human CRISP1.

Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1)

PubMed Central

Da Ros, Vanina G.; Maldera, Julieta A.; Willis, William D.; Cohen, Débora J.; Goulding, Eugenia H.; Gelman, Diego M.; Rubinstein, Marcelo; Eddy, Edward M.; Cuasnicu, Patricia S.

2008-01-01

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm-zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1−/− animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1−/− mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1−/− sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1+/+ and Crisp1−/− sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1−/− sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family. PMID:18571638

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

PubMed Central

Gibbs, Gerard M.; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J.; Martínez-López, Pablo; Luis de la Vega-Beltràn, José; Lo, Jennifer C. Y.; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K.

2011-01-01

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function. PMID:21482758

In vivo functional mapping of the conserved protein domains within murine Themis1.

PubMed

Zvezdova, Ekaterina; Lee, Jan; El-Khoury, Dalal; Barr, Valarie; Akpan, Itoro; Samelson, Lawrence; Love, Paul E

2014-09-01

Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

Molecular and bioinformatical characterization of a novel superfamily of cysteine-rich peptides from arthropods.

PubMed

Zeng, Xian-Chun; Nie, Yao; Luo, Xuesong; Wu, Shifen; Shi, Wanxia; Zhang, Lei; Liu, Yichen; Cao, Hanjun; Yang, Ye; Zhou, Jianping

2013-03-01

The full-length cDNA sequences of two novel cysteine-rich peptides (referred to as HsVx1 and MmKTx1) were obtained from scorpions. The two peptides represent a novel class of cysteine-rich peptides with a unique cysteine pattern. The genomic sequence of HsVx1 is composed of three exons interrupted by two introns that are localized in the mature peptide encoding region and inserted in phase 1 and phase 2, respectively. Such a genomic organization markedly differs from those of other peptides from scorpions described previously. Genome-wide search for the orthologs of HsVx1 identified 59 novel cysteine-rich peptides from arthropods. These peptides share a consistent cysteine pattern with HsVx1. Genomic comparison revealed extensive intron length differences and intronic number and position polymorphisms among the genes of these peptides. Further analysis identified 30 cases of intron sliding, 1 case of intron gain and 22 cases of intron loss occurred with the genes of the HsVx1 and HsVx1-like peptides. It is interesting to see that three HsVx1-like peptides XP_001658928, XP_001658929 and XP_001658930 were derived from a single gene (XP gene): the former two were generated from alternative splicing; the third one was encoded by a DNA region in the reverse complementary strand of the third intron of the XP gene. These findings strongly suggest that the genes of these cysteine-rich peptides were evolved by intron sliding, intron gain/loss, gene recombination and alternative splicing events in response to selective forces without changing their cysteine pattern. The evolution of these genes is dominated by intron sliding and intron loss. Copyright © 2012 Elsevier Inc. All rights reserved.

The proteins of Fusobacterium spp. involved in hydrogen sulfide production from L-cysteine.

PubMed

Basic, Amina; Blomqvist, Madeleine; Dahlén, Gunnar; Svensäter, Gunnel

2017-03-14

Hydrogen sulfide (H 2 S) is a toxic foul-smelling gas produced by subgingival biofilms in patients with periodontal disease and is suggested to be part of the pathogenesis of the disease. We studied the H 2 S-producing protein expression of bacterial strains associated with periodontal disease. Further, we examined the effect of a cysteine-rich growth environment on the synthesis of intracellular enzymes in F. nucleatum polymorphum ATCC 10953. The proteins were subjected to one-dimensional (1DE) and two-dimensional (2DE) gel electrophoresis An in-gel activity assay was used to detect the H 2 S-producing enzymes; Sulfide from H 2 S, produced by the enzymes in the gel, reacted with bismuth forming bismuth sulfide, illustrated as brown bands (1D) or spots (2D) in the gel. The discovered proteins were identified with liquid chromatography - tandem mass spectrometry (LC-MS/MS). Cysteine synthase and proteins involved in the production of the coenzyme pyridoxal 5'phosphate (that catalyzes the production of H 2 S) were frequently found among the discovered enzymes. Interestingly, a higher expression of H 2 S-producing enzymes was detected from bacteria incubated without cysteine prior to the experiment. Numerous enzymes, identified as cysteine synthase, were involved in the production of H 2 S from cysteine and the expression varied among Fusobacterium spp. and strains. No enzymes were detected with the in-gel activity assay among the other periodontitis-associated bacteria tested. The expression of the H 2 S-producing enzymes was dependent on environmental conditions such as cysteine concentration and pH but less dependent on the presence of serum and hemin.

Structural studies of human glioma pathogenesis-related protein 1

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

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu; Koski, Raymond A.; Bonafé, Nathalie

2011-10-01

Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structuresmore » of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.« less

Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains.

PubMed

Bäckström, Malin; Ambort, Daniel; Thomsson, Elisabeth; Johansson, Malin E V; Hansson, Gunnar C

2013-06-01

The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins.

Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

PubMed Central

Broer, Rene; Boson, Bertrand; Spaan, Willy; Cosset, François-Loïc; Corver, Jeroen

2006-01-01

The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity. PMID:16415007

Cysteine-Rich Intestinal Protein 1 Silencing Inhibits Migration and Invasion in Human Colorectal Cancer.

PubMed

He, Guoyang; Zou, Liyuan; Zhou, Lin; Gao, Peiqiong; Qian, Xinlai; Cui, Jing

2017-01-01

Cysteine-rich intestinal protein 1 (CRIP1), a member of the LIM/double zinc finger protein family, is abnormally expressed in several tumour types. However, few data are available on the role of CRIP1 in cancer. In the present study, we aimed to investigate the expression profile and functions of CRIP1 in colorectal cancer. To examine the protein expression level of CRIP1, immunohistochemistry (IHC) was performed on 56 pairs of colon cancer tissue samples. Western blotting was performed to investigate CRIP1 protein expression in four colon cancer cell lines. The endogenous expression of CRIP1 was suppressed using short interfering RNAs (siRNAs). Cell proliferation assays were used to determine whether CRIP1 silencing affected cell proliferation. Flow cytometry analysis was used to detect cell apoptosis. The effects of silencing CRIP1 on cell migration and invasion was detected using the transwell and wound-healing assays. IHC analysis showed that protein level of CRIP1 was significantly higher in tumour tissue samples than in paired non-tumour tissue samples and that the CRIP1 level was higher in metastatic tissue samples than in non-metastatic tissue samples. In addition, protein levels of CRIP1 were higher in highly metastatic colon cancer cell lines than in colon cancer cell lines with low metastasis. Further, CRIP1 silencing had no effect on cell proliferation or apoptosis in SW620 and HT29 cells. CRIP1 silencing suppressed cell migration and invasion obviously in SW620 and HT29 cells. The present study provides new evidence that abnormal expression of CRIP1 might be related to the degree of metastasis in colorectal cancer and that CRIP1 silencing could effectively inhibit migration and invasion during colorectal cancer development. These findings might aid the development of a biomarker for colon cancer prognosis and metastasis, and thus help to treat this common type of cancer. © 2017 The Author(s). Published by S. Karger AG, Basel.

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340*

PubMed Central

Loimaranta, Vuokko; Hytönen, Jukka; Pulliainen, Arto T.; Sharma, Ashu; Tenovuo, Jorma; Strömberg, Nicklas; Finne, Jukka

2009-01-01

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340. PMID:19465482

Human ESP1/CRP2, a member of the LIM domain protein family: Characterization of the cDNA and assignment of the gene locus to chromosome 14q32.3

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

Karim, Mohammad Azharul; Ohta, Kohji; Matsuda, Ichiro

1996-01-15

The LIM domain is present in a wide variety of proteins with diverse functions and exhibits characteristic arrangements of Cys and His residues with a novel zinc-binding motif. LIM domain proteins have been implicated in development, cell regulation, and cell structure. A LIM domain protein was identified by screening a human cDNA library with rat cysteine-rich intestinal protein (CRIP) as a probe, under conditions of low stringency. Comparison of the predicted amino acid sequence with several LIM domain proteins revealed 93% of the residues to be identical to rat LIM domain protein, termed ESP1 or CRP2. Thus, the protein ismore » hereafter referred to as human ESP1/CRP2. The cDNA encompasses a 1171-base region, including 26, 624, and 521 bases in the 5{prime}-noncoding region, coding region, and 3{prime}-noncoding regions, respectively, and encodes the entire ESP1/CRP2 protein has two LIM domains, and each shares 35.1% and 77 or 79% identical residues with human cysteine-rich protein (CRP) and rat CRIP, respectively. Northern blot analysis of ESP1/CRP2 in various human tissues showed distinct tissue distributions compared with CRP and CRIP, suggesting that each might serve related but specific roles in tissue organization or function. Using a panel of human-rodent somatic cell hybrids, the ESP1/CRP2 locus was assigned to chromosome 14. Fluorescence in situ hybridization, using cDNA and a genome DNA fragment of the ESP1/CRP2 as probes, confirms this assignment and relegates regional localization to band 14q32.3 47 refs., 7 figs.« less

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry.

PubMed

Mason, Alexander F; Thordarson, Pall

2016-07-20

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation. We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

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

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu

2011-05-01

The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structuremore » was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.« less

Molecular characterization of the equine testis-specific protein 1 (TPX1) and acidic epididymal glycoprotein 2 (AEG2) genes encoding members of the cysteine-rich secretory protein (CRISP) family.

PubMed

Giese, Alexander; Jude, Rony; Kuiper, Heidi; Raudsepp, Terje; Piumi, Francois; Schambony, Alexandra; Guérin, Gérard; Chowdhary, Bhanu P; Distl, Ottmar; Töpfer-Petersen, Edda; Leeb, Tosso

2002-10-16

The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 137 kb of horse genomic DNA from equine chromosome 20q22 containing the closely linked equine TPX1 and AEG2 genes are described. The equine TPX1 gene consists of ten exons spanning 18 kb while the AEG2 gene consists of eight exons that are spread over 24 kb. The expression of these two genes was investigated in several tissues by reverse transcription polymerase chain reaction analysis and Western blotting. Comparative genome analysis between horse, human, and mouse indicates that all three CRISP genes are clustered on one chromosomal location, which shows conserved synteny between these species.

Secreted protein acidic and rich in cysteine functions in colitis via IL17A regulation in mucosal CD4+ T cells.

PubMed

Tanaka, Makoto; Takagi, Tomohisa; Naito, Yuji; Uchiyama, Kazuhiko; Hotta, Yuma; Toyokawa, Yuki; Ushiroda, Chihiro; Hirai, Yasuko; Aoi, Wataru; Higashimura, Yasuki; Mizushima, Katsura; Okayama, Tetsuya; Katada, Kazuhiro; Kamada, Kazuhiro; Ishikawa, Takeshi; Handa, Osamu; Itoh, Yoshito

2018-03-01

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycol that regulates cell proliferation, tissue repair, and tumorigenesis. Despite evidence linking SPARC to inflammation, the mechanisms are unclear. Accordingly, the role of SPARC in intestinal inflammation was investigated. Colitis was induced in wild-type (WT) and SPARC knockout (KO) mice using trinitrobenzene sulfonic acid (TNBS). Colons were assessed for damage; leukocyte infiltration; Tnf, Ifng, Il17a, and Il10 mRNA expression; and histology. Cytokine profiling of colonic lamina propria mononuclear cells (LPMCs) was performed by flow cytometry. Naïve CD4 + T cells were isolated from WT and SPARC KO mouse spleens, and the effect of SPARC on Th17 cell differentiation was examined. Recombination activating gene 1 knockout (RAG1 KO) mice reconstituted with T cells from either WT or SPARC KO mice were investigated. Trinitrobenzene sulfonic acid exposure significantly reduced bodyweight and increased mucosal inflammation, leukocyte infiltration, and Il17a mRNA expression in WT relative to SPARC KO mice. The percentage of IL17A-producing CD4 + T cells among LPMCs from KO mice was lower than that in WT mice when both groups were exposed to TNBS. Th17 cell differentiation was suppressed in cells from SPARC KO mice. In the T cell transfer colitis model, RAG1 KO mice receiving T cells from WT mice were more severely affected than those reconstituted with cells from SPARC KO mice. Secreted protein acidic and rich in cysteine accelerates colonic mucosal inflammation via modulation of IL17A-producing CD4 + T cells. SPARC is a potential therapeutic target for conditions involving intestinal inflammation. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

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

Lovrinovic, Marina; Niemeyer, Christof M.

2005-09-30

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

PubMed

Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

2015-08-01

Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1

USDA-ARS?s Scientific Manuscript database

The gene encoding SnTox1, a necrotrophic effector from Stagonospora nodorum that causes necrosis of wheat lines expressing Snn1, has been verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid cysteine rich protein with the first 17 amino acids predicted as a signal ...

[Effects of redox state of disulfide bonds on the intrinsic fluorescence and denaturation of Trx-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea].

PubMed

Zhang, Teng; Feng, Juan; Li, Yang; Chen, Rui; Tang, Li-Xia; Pang, Xiao-Feng; Ren, Zheng-Long

2010-02-01

In the present paper, thioredoxin-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea, desigated as Trx-GcGASA and expressed prokaryotically, was purified and identified by using Ni(2+) -NTA affinity chromatography column and SDS-PAGE, and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol (DTT), oxidized glutathione (GSSG), peroxide and guanidine hydrochloride (GdnHCl) by means of steady-state fluorescence spectroscopic methods. It was found that (1) at the neutral pH Trx-GcGASA had maximum fluorescence emission at 305 nm following excitation at different wavelengths varying from 250 to 280 nm, which was ascribed to the fluorescence emission from tyrosine residues. (2) The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryptophan residues from 0.7 to 1.8. (3) Both Tyr and Trp residues underwent 12%-21% decrease in fluorescence intensity with the addition of 0.5 mmol x L(-1) GSSG or 5 mmol x L(-1) peroxide. The latter was roughly consistent with the antioxidative activity reported in vivo. (4) No matter whether 1 mmol x L(-1) DTT was absent or present, the fusion protein could not be fully unfolded with lambda(max) < 350 nm following the treatment of 6 mol x L(-1) GdnHCl. (5) Fusion protein Trx-GcGASA experienced GdnHCl-induced denaturation process, and the unfolding equilibrium curve could be well fitted by using two-state model, giving the Gibbs free energy change (deltaG) of 3.7 kJ x mol(-1). However, it was not the case for reduced Trx-GcGASA protein. The aforementioned experimental results will not only provide some guides to investigate the effects of fusion partner Trx on the unfolding thermodynamics, kinetics and refolding process of Trx-GcGASA, but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods.

Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

PubMed

Adade, Camila M; Carvalho, Ana Lúcia O; Tomaz, Marcelo A; Costa, Tatiana F R; Godinho, Joseane L; Melo, Paulo A; Lima, Ana Paula C A; Rodrigues, Juliany C F; Zingali, Russolina B; Souto-Padrón, Thaïs

2014-10-01

The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and Leishmania

PubMed Central

Adade, Camila M.; Carvalho, Ana Lúcia O.; Tomaz, Marcelo A.; Costa, Tatiana F. R.; Godinho, Joseane L.; Melo, Paulo A.; Lima, Ana Paula C. A.; Rodrigues, Juliany C. F.; Zingali, Russolina B.; Souto-Padrón, Thaïs

2014-01-01

Background The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Methodology/Principal Findings Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10–2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. Conclusions This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases. PMID:25330220

A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity.

PubMed

Isom, Daniel G; Marguet, Philippe R; Oas, Terrence G; Hellinga, Homme W

2011-04-01

Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics. Copyright © 2010 Wiley-Liss, Inc.

Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation

PubMed Central

Ymer, Susie I.; Greenall, Sameer A.; Cvrljevic, Anna; Cao, Diana X.; Donoghue, Jacqui F.; Epa, V. Chandana; Scott, Andrew M.; Adams, Timothy E.; Johns, Terrance G.

2011-01-01

The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation. PMID:24212795

Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematode Fasciola hepatica.

PubMed

Cancela, Martín; Corvo, Ileana; DA Silva, Edileuza; Teichmann, Aline; Roche, Leda; Díaz, Alvaro; Tort, José Fransisco; Ferreira, Henrique B; Zaha, Arnaldo

2017-11-01

Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.

Controlling the prion propensity of glutamine/asparagine-rich proteins.

PubMed

Paul, Kacy R; Ross, Eric D

2015-01-01

The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.

Overexpression of an Arabidopsis cysteine-rich receptor-like protein kinase, CRK5, enhances abscisic acid sensitivity and confers drought tolerance.

PubMed

Lu, Kai; Liang, Shan; Wu, Zhen; Bi, Chao; Yu, Yong-Tao; Wang, Xiao-Fang; Zhang, Da-Peng

2016-09-01

Receptor-like kinases (RLKs) have been reported to regulate many developmental and defense process, but only a few members have been functionally characterized. In the present study, our observations suggest that one of the RLKs, a membrane-localized cysteine-rich receptor-like protein kinase, CRK5, is involved in abscisic acid (ABA) signaling in Arabidopsis thaliana Overexpression of CRK5 increases ABA sensitivity in ABA-induced early seedling growth arrest and promotion of stomatal closure and inhibition of stomatal opening. Interestingly, and importantly, overexpression of CRK5 enhances plant drought tolerance without affecting plant growth at the mature stages and plant productivity. Transgenic lines overexpressing a mutated form of CRK5, CRK5 (K372E) with the change of the 372nd conserved amino acid residue from lysine to glutamic acid in its kinase domain, result in wild-type ABA and drought responses, supporting the role of CRK5 in ABA signaling. The loss-of-function mutation of the CRK5 gene does not affect the ABA response, while overexpression of two homologs of CRK5, CRK4 and CRK19, confers ABA responses, suggesting that these CRK members function redundantly. We further showed that WRKY18, WRKY40 and WRKY60 transcription factors repress the expression of CRK5, and that CRK5 likely functions upstream of ABI2 in ABA signaling. These findings help in understanding the complex ABA signaling network. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Impact of Secreted Protein Acidic and Rich in Cysteine (SPARC) Expression on Prognosis After Surgical Resection for Biliary Carcinoma.

PubMed

Toyota, Kazuhiro; Murakami, Yoshiaki; Kondo, Naru; Uemura, Kenichiro; Nakagawa, Naoya; Takahashi, Shinya; Sueda, Taijiro

2017-06-01

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that influences chemotherapy effectiveness and prognosis. The aim of this study was to investigate whether SPARC expression correlates with the postoperative survival of patients treated with surgical resection for biliary carcinoma. SPARC expression in resected biliary carcinoma specimens was investigated immunohistochemically in 175 patients. The relationship between SPARC expression and prognosis after surgery was evaluated using univariate and multivariate analyses. High SPARC expression in peritumoral stroma was found in 61 (35%) patients. In all patients, stromal SPARC expression was significantly associated with overall survival (OS) (P = 0.006). Multivariate analysis revealed that high stromal SPARC expression was an independent risk factor for poor OS (HR 1.81, P = 0.006). Moreover, high stromal SPARC expression was independently associated with poor prognosis in a subset of 118 patients treated with gemcitabine-based adjuvant chemotherapy (HR 2.04, P = 0.010) but not in the 57 patients who did not receive adjuvant chemotherapy (P = 0.21). Stromal SPARC expression correlated with the prognosis of patients with resectable biliary carcinoma, and its significance was enhanced in patients treated with adjuvant gemcitabine-based chemotherapy.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis

PubMed Central

Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J.

2016-01-01

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions. PMID:26771603

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

PubMed

Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J

2016-01-12

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

Controlling the prion propensity of glutamine/asparagine-rich proteins

PubMed Central

Paul, Kacy R; Ross, Eric D

2015-01-01

ABSTRACT The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve. PMID:26555096

Electrostatics of cysteine residues in proteins: Parameterization and validation of a simple model

PubMed Central

Salsbury, Freddie R.; Poole, Leslie B.; Fetrow, Jacquelyn S.

2013-01-01

One of the most popular and simple models for the calculation of pKas from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pKas. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pKas; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pKas. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pKa values (where the calculation should reproduce the pKa within experimental error). Both the general behavior of cysteines in proteins and the perturbed pKa in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pKa should be shifted, and validation of force field parameters for cysteine residues. PMID:22777874

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

PubMed

Oliveira, Marco A S; Baura, Valter A; Aquino, Bruno; Huergo, Luciano F; Kadowaki, Marco A S; Chubatsu, Leda S; Souza, Emanuel M; Dixon, Ray; Pedrosa, Fábio O; Wassem, Roseli; Monteiro, Rose A

2009-01-01

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins

PubMed Central

Yao, Chunxiang; Behring, Jessica B.; Shao, Di; Sverdlov, Aaron L.; Whelan, Stephen A.; Elezaby, Aly; Yin, Xiaoyan; Siwik, Deborah A.; Seta, Francesca; Costello, Catherine E.; Cohen, Richard A.; Matsui, Reiko; Colucci, Wilson S.; McComb, Mark E.; Bachschmid, Markus M.

2015-01-01

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation. PMID:26642319

Isolation of a new class of cysteine–glycine–proline-rich beta-proteins (beta-keratins) and their expression in snake epidermis

PubMed Central

Dalla Valle, Luisa; Nardi, Alessia; Alibardi, Lorenzo

2010-01-01

Scales of snakes contain hard proteins (beta-keratins), now referred to as keratin-associated beta-proteins. In the present study we report the isolation, sequencing, and expression of a new group of these proteins from snake epidermis, designated cysteine–glycine–proline-rich proteins. One deduced protein from expressed mRNAs contains 128 amino acids (12.5 kDa) with a theoretical pI at 7.95, containing 10.2% cysteine and 15.6% glycine. The sequences of two more snake cysteine–proline-rich proteins have been identified from genomic DNA. In situ hybridization shows that the messengers for these proteins are present in the suprabasal and early differentiating beta-cells of the renewing scale epidermis. The present study shows that snake scales, as previously seen in scales of lizards, contain cysteine-rich beta-proteins in addition to glycine-rich beta-proteins. These keratin-associated beta-proteins mix with intermediate filament keratins (alpha-keratins) to produce the resistant corneous layer of snake scales. The specific proportion of these two subfamilies of proteins in different scales can determine various degrees of hardness in scales. PMID:20070430

Electrostatics of cysteine residues in proteins: parameterization and validation of a simple model.

PubMed

Salsbury, Freddie R; Poole, Leslie B; Fetrow, Jacquelyn S

2012-11-01

One of the most popular and simple models for the calculation of pK(a) s from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pK(a) s. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pK(a) s; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pK(a) s. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pK(a) values (where the calculation should reproduce the pK(a) within experimental error). Both the general behavior of cysteines in proteins and the perturbed pK(a) in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pK(a) should be shifted, and validation of force field parameters for cysteine residues. Copyright © 2012 Wiley Periodicals, Inc.

The CUG-initiated larger form coat protein of Chinese wheat mosaic virus binds to the cysteine-rich RNA silencing suppressor.

PubMed

Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Ratti, Claudio; Chen, Jianping

2013-10-01

Some viruses use alternative translation initiation at non-AUG codons as a strategy to produce multiple proteins during gene expression. Here we show that, using this strategy, Chinese wheat mosaic virus (CWMV; Furovirus) expresses a larger form of coat protein (N-ext/CP) in infected plants. Site-directed mutagenesis and transient expression analysis confirmed that CWMV N-ext/CP is initiated at an upstream in-frame CUG codon at nucleotide position 207-209 of RNA 2, which adds a 39 amino acid (aa) N-terminal extension to the major CP. Interestingly, in planta and in vitro analyses indicated that CWMV N-ext/CP but not CP interacts with the CWMV cysteine-rich protein (CRP), an RNA silencing suppressor. We further determined that the N-terminal 39 aa extension, particularly the 10 aa region immediately upstream of the major CP coding region is responsible for the interaction of N-ext/CP with CRP. In an Agrobacterium co-infiltration assay, co-expression with N-ext/CP did not affect CRP silencing suppression activity. Thus the alternative translation initiation at a CUG codon provides the CWMV N-ext/CP with the ability to bind to the viral silencing suppressor. Copyright © 2013 Elsevier B.V. All rights reserved.

Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

PubMed

Wible, Ryan S; Sutter, Thomas R

2017-03-20

The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

Detection of Cysteine Redox States in Mitochondrial Proteins in Intact Mammalian Cells.

PubMed

Habich, Markus; Riemer, Jan

2017-01-01

Import, folding, and activity regulation of mitochondrial proteins are important for mitochondrial function. Cysteine residues play crucial roles in these processes as their thiol groups can undergo (reversible) oxidation reactions. For example, during import of many intermembrane space (IMS) proteins, cysteine oxidation drives protein folding and translocation over the outer membrane. Mature mitochondrial proteins can undergo changes in the redox state of specific cysteine residues, for example, as part of their enzymatic reaction cycle or as adaptations to changes of the local redox environment which might influence their activity. Here we describe methods to study changes in cysteine residue redox states in intact cells. These approaches allow to monitor oxidation-driven protein import as well as changes of cysteine redox states in mature proteins during oxidative stress or during the reaction cycle of thiol-dependent enzymes like oxidoreductases.

Efficient Site-Specific Labeling of Proteins via Cysteines

PubMed Central

Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

2011-01-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

Efficient site-specific labeling of proteins via cysteines.

PubMed

Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

2008-03-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

Molecular mechanisms involved in gamete interaction: evidence for the participation of cysteine-rich secretory proteins (CRISP) in sperm-egg fusion.

PubMed

Da Ros, V; Busso, D; Cohen, D J; Maldera, J; Goldweic, N; Cuasnicu, P S

2007-01-01

Epididymal protein DE and testicular protein Tpx-1 are two cysteine-rich secretory proteins also known as CRISP-1 and CRISP-2, respectively. DE/ CRISP-1 is localised on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites. Recent results using bacterially-expressed recombinant fragments of DE as well as synthetic peptides revealed that the ability of DE to bind to the egg surface and inhibit gamete fusion resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family (Signature 2). Given the high degree of homology between DE/CRISP-1 and Tpx-1/CRISP-2, we also explored the potential participation of the testicular intra-acrosomal protein in gamete fusion. Results showing the ability of recombinant Tpx-1 to bind to the surface of rat eggs (evaluated by indirect immunofluorescence) and to significantly inhibit zona-free egg penetration, support the participation of this protein in gamete fusion through its interaction with egg-binding sites. Interestingly, rat Tpx-1 exhibits only two substitutions in Signature 2 when compared to this region in DE. Together, these results provide evidence for the involvement of both epididymal DE/CRISP-1 and testicular Tpx-1/CRISP-2 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilisation.

Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

PubMed

Lipton, Stuart A; Choi, Yun-Beom; Takahashi, Hiroto; Zhang, Dongxian; Li, Weizhong; Godzik, Adam; Bankston, Laurie A

2002-09-01

Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the same thiol group on a single cysteine residue, forming a molecular switch composed of a latticework of possible redox, NO or Zn(2+) modifications to control protein function. Thiol-mediated regulation of protein function can also involve reactions of cysteine residues that affect ligand binding allosterically. This article reviews the basis for these molecular cysteine switches, drawing on the NMDA receptor as an exemplary protein, and proposes a molecular model for the action of S-nitrosylation based on recently derived crystal structures.

Preferential expression of cystein-rich secretory protein-3 (CRISP-3) in chronic pancreatitis.

PubMed

Liao, Q; Kleeff, J; Xiao, Y; Guweidhi, A; Schambony, A; Töpfer-Petersen, E; Zimmermann, A; Büchler, M W; Friess, H

2003-04-01

Chronic pancreatitis (CP) is a progressive inflammatory process resulting in exocrine and endocrine pancreatic insufficiency in advanced stages. Cysteine-rich secretory protein (CRISP-3) has been identified as a defense-associated molecule with predominant expression in the salivary gland, pancreas and prostate. In this study, we investigated CRISP-3 expression in normal pancreatic tissues, chronic pancreatitis tissues, pancreatic cancer tissues and pancreatic cancer cell lines, as well as in other gastrointestinal organs. 15 normal pancreatic tissues, 14 chronic pancreatitis tissues and 14 pancreatic cancer tissues as well as three pancreatic cancer cell lines were analyzed. Moreover, hepatocellular carcinoma and esophageal, stomach and colon cancers were also analyzed together with the corresponding normal controls. CRISP-3 was expressed at moderate to high levels in chronic pancreatitis tissues and at moderate levels in pancreatic cancer tissues but at low levels in normal pancreatic tissues, and was absent in three pancreatic cancer cell lines. CRISP-3 expression was below the level of detection in all cancerous gastrointestinal tissues and in all normal tissues except 2 of 16 colon tissue samples. CRISP-3 mRNA signals and immunoreactivity were strongly present in the cytoplasm of degenerating acinar cells and in small proliferating ductal cells in CP tissues and CP-like lesions in pancreatic cancer tissues. In contrast, CRISP-3 expression was weak to absent in the cytoplasm of cancer cells as well as in acinar cells and ductal cells in pancreatic cancer tissues and normal pancreatic tissues. These results reveal that the distribution of CRISP-3 in gastrointestinal tissues is predominantly in the pancreas. High levels of CRISP-3 in acinar cells dedifferentiating into small proliferating ductal cells in CP and CP-like lesions in pancreatic cancer suggests a role of this molecule in the pathophysiology of CP.

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

PubMed

Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

2012-06-01

Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

Side chain-side chain interactions of arginine with tyrosine and aspartic acid in Arg/Gly/Tyr-rich domains within plant glycine-rich RNA binding proteins.

PubMed

Kumaki, Yasuhiro; Nitta, Katsutoshi; Hikichi, Kunio; Matsumoto, Takeshi; Matsushima, Norio

2004-07-01

Plant glycine-rich RNA-binding proteins (GRRBPs) contain a glycine-rich region at the C-terminus whose structure is quite unknown. The C-terminal glycine-rich part is interposed with arginine and tyrosine (arginine/glycine/tyrosine (RGY)-rich domain). Comparative sequence analysis of forty-one GRRBPs revealed that the RGY-rich domain contains multiple repeats of Tyr-(Xaa)h-(Arg)k-(Xaa)l, where Xaa is mainly Gly, "k" is 1 or 2, and "h" and "l" range from 0 to 10. Two peptides, 1 (G1G2Y3G4G5G6R7R8D9G10) and 2 (G1G2R3R4D5G6G7Y8G9G10), corresponding to sections of the RGY-rich domain in Zea mays RAB15, were selected for CD and NMR experiments. The CD spectra indicate a unique, positive band near 228 nm in both peptides that has been ascribed to tyrosine residues in ordered structures. The pH titration by NMR revealed that a side chain-side chain interaction, presumably an H-Nepsilon...O=Cgamma hydrogen bonding interaction in the salt bridge, occurs between Arg (i) and Asp (i + 2). 1D GOESY experiments indicated the presence of NOE between the aromatic side chain proton and the arginine side chain proton in the two peptides suggesting strongly that the Arg (i) aromatic side chain interacts directly with the Tyr (i +/- 4 or i +/- 5) side chain.

Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance

PubMed Central

Ortega-Amaro, María A.; Rodríguez-Hernández, Aída A.; Rodríguez-Kessler, Margarita; Hernández-Lucero, Eloísa; Rosales-Mendoza, Sergio; Ibáñez-Salazar, Alejandro; Delgado-Sánchez, Pablo; Jiménez-Bremont, Juan F.

2015-01-01

Proteins with glycine-rich signatures have been reported in a wide variety of organisms including plants, mammalians, fungi, and bacteria. Plant glycine-rich protein genes exhibit developmental and tissue-specific expression patterns. Herein, we present the characterization of the AtGRDP2 gene using Arabidopsis null and knockdown mutants and, Arabidopsis and lettuce over-expression lines. AtGRDP2 encodes a short glycine-rich domain protein, containing a DUF1399 domain and a putative RNA recognition motif (RRM). AtGRDP2 transcript is mainly expressed in Arabidopsis floral organs, and its deregulation in Arabidopsis Atgrdp2 mutants and 35S::AtGRDP2 over-expression lines produces alterations in development. The 35S::AtGRDP2 over-expression lines grow faster than the WT, while the Atgrdp2 mutants have a delay in growth and development. The over-expression lines accumulate higher levels of indole-3-acetic acid and, have alterations in the expression pattern of ARF6, ARF8, and miR167 regulators of floral development and auxin signaling. Under salt stress conditions, 35S::AtGRDP2 over-expression lines displayed higher tolerance and increased expression of stress marker genes. Likewise, transgenic lettuce plants over-expressing the AtGRDP2 gene manifest increased growth rate and early flowering time. Our data reveal an important role for AtGRDP2 in Arabidopsis development and stress response, and suggest a connection between AtGRDP2 and auxin signaling. PMID:25653657

Investigating possible biological targets of Bj-CRP, the first cysteine-rich secretory protein (CRISP) isolated from Bothrops jararaca snake venom.

PubMed

Lodovicho, Marina E; Costa, Tássia R; Bernardes, Carolina P; Menaldo, Danilo L; Zoccal, Karina F; Carone, Sante E; Rosa, José C; Pucca, Manuela B; Cerni, Felipe A; Arantes, Eliane C; Tytgat, Jan; Faccioli, Lúcia H; Pereira-Crott, Luciana S; Sampaio, Suely V

2017-01-04

Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis.

PubMed

Cabrillana, María E; Monclus, María A; Sáez Lancellotti, Tania E; Boarelli, Paola V; Clementi, Marisa A; Vincenti, Amanda E; Yunes, Roberto F M; Fornés, Miguel W

2011-09-01

Mammalian sperm proteins undergo thiol group (SH) oxidation to form disulfides bonds (SS) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelle stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in the head, but also in the tail, indicates that both sub cellular regions participate in the thiol changes. The sources of the fluorescence signal are sulfhydryls sperm proteins labeled by mBBr (mBBr-spp). Initial attempts to identify the mBBr-spp labeled were detected in the initial-caput, but not in the distal cauda-segment of the epididymis in sodium dodecyl sulfate (SDS)-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason, disulfide bond reduction was accomplished by sodium dodecyl sulfate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band displays a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). The mBBr specifically bound to N-terminal domain cysteine of ODF1. The mBBr reduces rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significantly increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in the sperm tail, and the motility inhibition by the fluorescence marker indicates that ODF1 N-terminal domain are related to sperm motility. © 2011 Wiley-Liss, Inc. Copyright © 2011 Wiley-Liss, Inc.

Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein.

PubMed

Candel, Adela M; van Nuland, Nico A J; Martin-Sierra, Francisco M; Martinez, Jose C; Conejero-Lara, Francisco

2008-03-14

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the alpha-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the "unbound" and "bound" states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2 approximately P7 approximately P10>P9 approximately P6>P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy

Identification of human cysteine-rich secretory protein 3 (CRISP-3) as a matrix protein in a subset of peroxidase-negative granules of neutrophils and in the granules of eosinophils.

PubMed

Udby, Lene; Calafat, Jero; Sørensen, Ole E; Borregaard, Niels; Kjeldsen, Lars

2002-09-01

Cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) was originally discovered in human neutrophilic granulocytes. We have recently developed a sensitive sandwich enzyme-linked immunosorbent assay for CRISP-3 and demonstrated the presence of CRISP-3 in exocrine secretions. To investigate the subcellular localization and mobilization of CRISP-3 in human neutrophils, we performed subcellular fractionation of resting and activated neutrophils on three-layer Percoll density gradients, release-studies of granule proteins in response to different secretagogues, and double-labeling immunogold electron microscopy. CRISP-3 was found to be localized in a subset of granules with overlapping characteristics of specific and gelatinase granules and mobilized accordingly, thus confirming the hypothesis that peroxidase-negative granules exist as a continuum from specific to gelatinase granules regarding protein content and mobilization. CRISP-3 was found to be a matrix protein, which is stored in granules as glycosylated and as unglycosylated protein. The subcellular distribution of the two forms of CRISP-3 was identical. In addition, CRISP-3 was found as a granule protein in eosinophilic granulocytes. The presence of CRISP-3 in peroxidase-negative granules of neutrophils, in granules of eosinophils, and in exocrine secretions indicates a role in the innate host defense.

EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.

PubMed

Pearson, Jaclyn S; Giogha, Cristina; Mühlen, Sabrina; Nachbur, Ueli; Pham, Chi L L; Zhang, Ying; Hildebrand, Joanne M; Oates, Clare V; Lung, Tania Wong Fok; Ingle, Danielle; Dagley, Laura F; Bankovacki, Aleksandra; Petrie, Emma J; Schroeder, Gunnar N; Crepin, Valerie F; Frankel, Gad; Masters, Seth L; Vince, James; Murphy, James M; Sunde, Margaret; Webb, Andrew I; Silke, John; Hartland, Elizabeth L

2017-01-13

Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling 1-3 . RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis 4,5 . Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides

PubMed Central

Yoga, Yano M. K.; Traore, Daouda A. K.; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R.; Barker, Andrew; Leedman, Peter J.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

2012-01-01

Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5′-CCCTCCCT-3′ DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5′-ACCCCA-3′ DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad. PMID:22344691

Mapping of the local environmental changes in proteins by cysteine scanning

PubMed Central

Yamazaki, Yoichi; Nagata, Tomoko; Terakita, Akihisa; Kandori, Hideki; Shichida, Yoshinori; Imamoto, Yasushi

2014-01-01

Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes. PMID:27493492

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

PubMed

Prakobphol, A; Xu, F; Hoang, V M; Larsson, T; Bergstrom, J; Johansson, I; Frängsmyr, L; Holmskov, U; Leffler, H; Nilsson, C; Borén, T; Wright, J R; Strömberg, N; Fisher, S J

2000-12-22

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance

PubMed Central

Mulinta, Ras; Yao, Sylvia Y. M.; Ng, Amy M. L.; Cass, Carol E.; Young, James D.

2017-01-01

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function. PMID:28385889

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

PubMed Central

Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius

2014-01-01

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

Carbon Nanotubes Facilitate Oxidation of Cysteine Residues of Proteins.

PubMed

Hirano, Atsushi; Kameda, Tomoshi; Wada, Momoyo; Tanaka, Takeshi; Kataura, Hiromichi

2017-10-19

The adsorption of proteins onto nanoparticles such as carbon nanotubes (CNTs) governs the early stages of nanoparticle uptake into biological systems. Previous studies regarding these adsorption processes have primarily focused on the physical interactions between proteins and nanoparticles. In this study, using reduced lysozyme and intact human serum albumin in aqueous solutions, we demonstrated that CNTs interact chemically with proteins. The CNTs induce the oxidation of cysteine residues of the proteins, which is accounted for by charge transfer from the sulfhydryl groups of the cysteine residues to the CNTs. The redox reaction simultaneously suppresses the intermolecular association of proteins via disulfide bonds. These results suggest that CNTs can affect the folding and oxidation degree of proteins in biological systems such as blood and cytosol.

Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein

NASA Astrophysics Data System (ADS)

Darwiche, Rabih; Kelleher, Alan; Hudspeth, Elissa M.; Schneiter, Roger; Asojo, Oluwatoyin A.

2016-06-01

The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1.

Heterologous Gln/Asn-Rich Proteins Impede the Propagation of Yeast Prions by Altering Chaperone Availability

PubMed Central

Yang, Zi; Hong, Joo Y.; Derkatch, Irina L.; Liebman, Susan W.

2013-01-01

Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI +] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI +] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI +]. PMID:23358669

Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

PubMed Central

Stephens, Robert F.; Guan, W.; Zhorov, Boris S.; Spafford, J. David

2015-01-01

How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel. PMID

New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

PubMed

Jung, Woongsic; Campbell, Robert L; Gwak, Yunho; Kim, Jong Im; Davies, Peter L; Jin, EonSeon

2016-01-01

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

PubMed Central

Jung, Woongsic; Gwak, Yunho; Kim, Jong Im; Davies, Peter L.; Jin, EonSeon

2016-01-01

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP’s biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP. PMID:27097164

Selective Loss of Cysteine Residues and Disulphide Bonds in a Potato Proteinase Inhibitor II Family

PubMed Central

Li, Xiu-Qing; Zhang, Tieling; Donnelly, Danielle

2011-01-01

Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution. PMID:21494600

Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX.

PubMed

Argentaro, Anthony; Yang, Ji-Chun; Chapman, Lynda; Kowalczyk, Monika S; Gibbons, Richard J; Higgs, Douglas R; Neuhaus, David; Rhodes, Daniela

2007-07-17

The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with alpha-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal alpha-helix that pack together to form a single globular domain. Interestingly, the alpha-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.

Role of chirality in peptide-induced formation of cholesterol-rich domains

PubMed Central

2005-01-01

The chiral specificity of the interactions of peptides that induce the formation of cholesterol-rich domains has not been extensively investigated. Both the peptide and most lipids are chiral, so there is a possibility that interactions between peptide and lipid could require chiral recognition. On the other hand, in our models with small peptides, the extent of folding of the peptide to form a specific binding pocket is limited. We have determined that replacing cholesterol with its enantiomer, ent-cholesterol, alters the modulation of lipid organization by peptides. The phase-transition properties of SOPC (1-stearoyl-2-oleoylphosphatidylcholine):cholesterol [in a 6:4 ratio with 0.2 mol% PtdIns(4,5)P2] are not significantly altered when ent-cholesterol replaces cholesterol. However, in the presence of 10 mol% of a 19-amino-acid, N-terminally myristoylated fragment (myristoyl-GGKLSKKKKGYNVNDEKAK-amide) of the protein NAP-22 (neuronal axonal membrane protein), the lipid mixture containing cholesterol undergoes separation into cholesterol-rich and cholesterol-depleted domains. This does not occur when ent-cholesterol replaces cholesterol. In another example, when N-acetyl-Leu-Trp-Tyr-Ile-Lys-amide (N-acetyl-LWYIK-amide) is added to SOPC:cholesterol (7:3 ratio), there is a marked increase in the transition enthalpy of the phospholipid, indicating separation of a cholesterol-depleted domain of SOPC. This phenomenon completely disappears when ent-cholesterol replaces cholesterol. The all-D-isomer of N-acetyl-LWYIK-amide also induces the formation of cholesterol-rich domains with natural cholesterol, but does so to a lesser extent with ent-cholesterol. Thus specific peptide chirality is not required for interaction with cholesterol-containing membranes. However, a specific chirality of membrane lipids is required for peptide-induced formation of cholesterol-rich domains. PMID:15929726

Structure of the substrate-binding b′ domain of the Protein disulfide isomerase-like protein of the testis

PubMed Central

Bastos-Aristizabal, Sara; Kozlov, Guennadi; Gehring, Kalle

2014-01-01

Protein Disulfide Isomerase-Like protein of the Testis (PDILT) is a testis-specific member of the PDI family. PDILT displays similar domain architecture to PDIA1, the founding member of this protein family, but lacks catalytic cysteines needed for oxidoreduction reactions. This suggests special importance of chaperone activity of PDILT, but how it recognizes misfolded protein substrates is unknown. Here, we report the high-resolution crystal structure of the b′ domain of human PDILT. The structure reveals a conserved hydrophobic pocket, which is likely a principal substrate-binding site in PDILT. In the crystal, this pocket is occupied by side chains of tyrosine and tryptophan residues from another PDILT molecule, suggesting a preference for binding exposed aromatic residues in protein substrates. The lack of interaction of the b′ domain with the P-domains of calreticulin-3 and calmegin hints at a novel way of interaction between testis-specific lectin chaperones and PDILT. Further studies of this recently discovered PDI member would help to understand the important role that PDILT plays in the differentiation and maturation of spermatozoids. PMID:24662985

The SH3 and cysteine-rich domain 3 (Stac3) gene is important to growth, fiber composition, and calcium release from the sarcoplasmic reticulum in postnatal skeletal muscle.

PubMed

Cong, Xiaofei; Doering, Jonathan; Mazala, Davi A G; Chin, Eva R; Grange, Robert W; Jiang, Honglin

2016-01-01

The SH3 and cysteine-rich domain 3 (Stac3) gene is specifically expressed in the skeletal muscle. Stac3 knockout mice die perinatally. In this study, we determined the potential role of Stac3 in postnatal skeletal muscle growth, fiber composition, and contraction by generating conditional Stac3 knockout mice. We disrupted the Stac3 gene in 4-week-old male mice using the Flp-FRT and tamoxifen-inducible Cre-loxP systems. RT-qPCR and western blotting analyses of the limb muscles of target mice indicated that nearly all Stac3 mRNA and more than 70 % of STAC3 protein were deleted 4 weeks after tamoxifen injection. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei, with no effect on the total myofiber number. Grip strength and grip time tests indicated that postnatal Stac3 deletion decreased limb muscle strength in mice. Muscle contractile tests revealed that postnatal Stac3 deletion reduced electrostimulation-induced but not the ryanodine receptor agonist caffeine-induced maximal force output in the limb muscles. Calcium imaging analysis of single flexor digitorum brevis myofibers indicated that postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. This study demonstrates that STAC3 is important to myofiber hypertrophy, myofiber-type composition, contraction, and excitation-induced calcium release from the sarcoplasmic reticulum in the postnatal skeletal muscle.

Two COWP-like cysteine rich proteins from Eimeria nieschulzi (coccidia, apicomplexa) are expressed during sporulation and involved in the sporocyst wall formation.

PubMed

Jonscher, Ernst; Erdbeer, Alexander; Günther, Marie; Kurth, Michael

2015-07-25

The family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can also be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well. Genome sequence analysis of Eimeria suggests that these proteins may also exist in this genus and led us to the assumption that these proteins may also play a role in oocyst wall formation. In this study, COWP-like encoding sequences had been identified in Eimeria nieschulzi. The predicted gene sequences were subsequently utilized in reporter gene assays to observe time of expression and localisation of the reporter protein in vivo. Both investigated proteins, EnOWP2 and EnOWP6, were expressed during sporulation. The EnOWP2-promoter driven mCherry was found in the cytoplasm and the EnOWP2, respectively EnOWP6, fused to mCherry was initially observed in the extracytoplasmatic space between sporoblast and oocyst wall. This, so far unnamed compartment was designated as circumplasm. Later, the mCherry reporter co-localised with the sporocyst wall of the sporulated oocysts. This observation had been confirmed by confocal microscopy, excystation experiments and IFA. Transcript analysis revealed the intron-exon structure of these genes and confirmed the expression of EnOWP2 and EnOWP6 during sporogony. Our results allow us to assume a role, of both investigated EnOWP proteins, in the sporocyst wall formation of E. nieschulzi. Data mining and sequence comparisons to T. gondii and other Eimeria species allow us to hypothesise a conserved process within the coccidia. A role in oocyst wall formation had not been observed in E. nieschulzi.

Domain architectures of the Scm3p protein provide insights into centromere function and evolution.

PubMed

Aravind, L; Iyer, Lakshminarayan M; Wu, Carl

2007-10-15

Recently, Scm3p has been shown to be a nonhistone component of centromeric chromatin that binds stoichiometrically to CenH3-H4 histones, and to be required for the assembly of kinetochores in Saccharomyces cerevisiae. Scm3p is conserved across fungi, and displays a remarkable variation in protein size, ranging from approximately 200 amino acids in S. cerevisiae to approximately 1300 amino acids in Neurospora crassa. This is primarily due a variable C-terminal segment that is linked to a conserved N-terminal, CenH3-interacting domain. We have discovered that the extended C-terminal region of Scm3p is strikingly characterized by lineage-specific fusions of single or multiple predicted DNA-binding domains different versions of the MYB and C2H2 zinc finger domains, AT-hooks, and a novel cysteine-rich metal-chelating cluster that are absent from the small versions of Scm3. Instead, S. cerevisiae point centromeres are recognized by components of the CBF3 DNA binding complex, which are conserved amongst close relatives of budding yeast, but are correspondingly absent from more distant fungi that possess regional centromeres. Hence, the C-terminal DNA binding motifs found in large Scm3p proteins may, along with CenH3, serve as a key epigenetic signal by recognizing and accommodating the lineage-specific diversity of centromere DNA in course of evolution.

Alternative splicing for members of human mosaic domain superfamilies. I. The CH and LIM domains containing group of proteins.

PubMed

Friedberg, Felix

2009-05-01

In this paper we examine (restricted to homo sapiens) the products resulting from gene duplication and the subsequent alternative splicing for the members of a multidomain group of proteins which possess the evolutionary conserved calponin homology CH domain, i.e. an "actin binding domain", as a singlet and which, in addition, contain the conserved cysteine rich double Zn finger possessing Lim domain, also as a singlet. Seven genes, resulting from gene duplications, were identified that code for seven group members for which pre-mRNAs appear to have undergone multiple alternative splicing: Mical 1, 2 and 3 are located on chromosomes 6q21, 11p15 and 22q11, respectively. The LMO7 gene is present on chromosome 13q22 and the LIMCH1 gene on chromosome 4p13. Micall1 is mapped to chromosome 22q13 and Micall2 to chromosome 7p22. Translated Gen/Bank ESTs suggest the existence of multiple products alternatively spliced from the pre-mRNAs encoded by these genes. Characteristic indicators of such splicing among the proteins derived from one gene must include containment of some common extensive 100% identical regions. In some instances only one exon might be partly or completely eliminated. Sometimes alternative splicing is also associated with an increased frequency of creation of an exon or part of an exon from an intron. Not only coding regions for the body of the protein but also for its N- or -C ends could be affected by the splicing. If created forms are merely beginning at different starting points but remain identical in sequence thereafter, their existence as products of alternate splicing must be questioned. In the splicings, described in this paper, multiple isoforms rather than a single isoform appear as products during the gene expression.

Binding of the Extracellular Eight-Cysteine Motif of Opy2 to the Putative Osmosensor Msb2 Is Essential for Activation of the Yeast High-Osmolarity Glycerol Pathway

PubMed Central

Yamamoto, Katsuyoshi

2015-01-01

To adapt to environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase, which regulates diverse osmoadaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of independent upstream signaling routes termed the SLN1 branch and the SHO1 branch. Here, we report that the extracellular cysteine-rich (CR) domain of the transmembrane-anchor protein Opy2 binds to the Hkr1-Msb2 homology (HMH) domain of the putative osmosensor Msb2 and that formation of the Opy2-Msb2 complex is essential for osmotic activation of Hog1 through the MSB2 subbranch of the SHO1 branch. By analyzing the phenotypes of mutants with Opy2 cysteine-to-alanine mutations, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for the potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed mutants with a site-specific Cys-to-Ala mutation of the Opy2 CR domain and mutants with a Cys substitution of the Msb2 HMH domain. Each of these mutants had a reduced cysteine. These mutants were then combinatorially cross-linked using chemical cross-linkers of different lengths. Cross-linking between Opy2 Cys48 and Msb2 Cys1023 was sensitive to osmotic changes, suggesting that osmostress induced a conformational change. We therefore propose that the Opy2-Msb2 complex might serve as an osmosensor. PMID:26598606

Cysteine-rich peptides (CRPs) mediate diverse aspects of cell-cell communication in plant reproduction and development.

PubMed

Marshall, Eleanor; Costa, Liliana M; Gutierrez-Marcos, Jose

2011-03-01

Cell-cell communication in plants is essential for the correct co-ordination of reproduction, growth, and development. Studies to dissect this mode of communication have previously focussed primarily on the action of plant hormones as mediators of intercellular signalling. In animals, peptide signalling is a well-documented intercellular communication system, however, relatively little is known about this system in plants. In recent years, numerous reports have emerged about small, secreted peptides controlling different aspects of plant reproduction. Interestingly, most of these peptides are cysteine-rich, and there is convincing evidence suggesting multiple roles for related cysteine-rich peptides (CRPs) as signalling factors in developmental patterning as well as during plant pathogen responses and symbiosis. In this review, we discuss how CRPs are emerging as key signalling factors in regulating multiple aspects of vegetative growth and reproductive development in plants.

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

PubMed

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-11-04

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

Cysteine-rich secretory protein 3 plays a role in prostate cancer cell invasion and affects expression of PSA and ANXA1.

PubMed

Pathak, Bhakti R; Breed, Ananya A; Apte, Snehal; Acharya, Kshitish; Mahale, Smita D

2016-01-01

Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

PubMed Central

Létoffé, S; Wandersman, C

1992-01-01

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

The Cysteine-Rich Domain of the Macrophage Mannose Receptor Is a Multispecific Lectin That Recognizes Chondroitin Sulfates a and B and Sulfated Oligosaccharides of Blood Group Lewisa and Lewisx Types in Addition to the Sulfated N-Glycans of Lutropin

PubMed Central

Leteux, Christine; Chai, Wengang; Loveless, R. Wendy; Yuen, Chun-Ting; Uhlin-Hansen, Lars; Combarnous, Yves; Jankovic, Mila; Maric, Svetlana C.; Misulovin, Ziva; Nussenzweig, Michel C.; Ten Feizi

2000-01-01

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells. PMID:10748230

Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide binding.

PubMed

Visperas, Patrick R; Winger, Jonathan A; Horton, Timothy M; Shah, Neel H; Aum, Diane J; Tao, Alyssa; Barros, Tiago; Yan, Qingrong; Wilson, Christopher G; Arkin, Michelle R; Weiss, Arthur; Kuriyan, John

2015-01-01

Zeta-chain associated protein of 70 kDa (ZAP-70) and spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signalling respectively. They are recruited, via their tandem-SH2 (Src-homology domain 2) domains, to doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signalling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of H2O2 and these two cysteine residues are also necessary for inhibition by H2O2. Our findings suggest a mechanism by which the reactive oxygen species generated during responses to antigen could attenuate signalling through these kinases and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.

MicroRNA-15b regulates reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression in human uterine leiomyoma.

PubMed

Guan, Yichun; Guo, Lankai; Zukerberg, Lawrence; Rueda, Bo R; Styer, Aaron K

2016-08-17

Human uterine leiomyoma (fibroids; LYO) are the most common benign neoplasms in reproductive-aged women. Dysregulated extracellular matrix and irregular LYO reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression are thought to be mediated by aberrant microRNA (miR) expression. The relationship of miR-15b and RECK expression in LYO has not been studied. The expression levels of miR-15b and RECK were determined by quantitative RT-PCR, Western blot, and immunohistochemistry in cultures derived from commercial primary leiomyoma (cpLYO) and myometrial (cpMYO) cell lines and leiomyoma (pLYO) and myometrium (pMYO) tissue from surgical samples respectively. The relationship between miR-15b and RECK expression in cpLYO and pLYO (compared to their respective myometrial controls) was evaluated following transfection of cell cultures with either miR-15b mimic or inhibitor. Elevated levels of miR-15b were observed in cpLYO (2.82-fold; p = 0.04) and pLYO cell (1.30-fold; p = 0.0001) cultures respectively compared to corresponding MYO cell controls. Following transfection with miR-15b mimic, cpLYO cells (0.62-fold; p < 0.0001) and pLYO cells (0.68-fold; p < 0.0001) demonstrated reduced RECK protein expression. Following transfection with miR-15b inhibitor, cpLYO cells (1.20-fold; p < 0.0001) and pLYO cells (1.31-fold; p = 0.0007) demonstrated elevated RECK protein expression. RECK protein expression was reduced in pLYO tissues (0.73-fold; p < 0.0001) and pLYO (0.47-fold; p = 0.047) cells when compared to the corresponding MYO tissue controls. Our findings suggest that miR-15b negatively regulates RECK expression in LYO, and increased miR-15b and decreased RECK expression may contribute to the pathobiology of LYO. The functional significance of miR-15b and RECK expression warrants further investigation as potential therapeutic targets for the treatment of human LYO.

Desulfurization of Cysteine-Containing Peptides Resulting from Sample Preparation for Protein Characterization by MS

PubMed Central

Wang, Zhouxi; Rejtar, Tomas; Zhou, Zhaohui Sunny; Karger, Barry L.

2010-01-01

In this paper, we have examined two cysteine modifications resulting from sample preparation for protein characterization by MS: (1) a previously observed conversion of cysteine to dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine to alanine. Using model peptides, the conversion of cysteine to dehydroalanine via β-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37 °C, pH 7.0– 9.0) without disulfide reduction and alkylation.. Furthermore, the surprising conversion of cysteine to alanine was shown to occur by heating cysteine containing peptides in the presence of a phosphine (TCEP). The formation of alanine from cysteine, investigated by performing experiments in H2O or D2O, suggested a radical-based desulfurization mechanism unrelated to β-elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. PMID:20049891

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome*

PubMed Central

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-01-01

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome. PMID:21917930

Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.

PubMed

Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

2013-11-01

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions

PubMed Central

Francin-Allami, Mathilde; Saumonneau, Amélie; Lavenant, Laurence; Bouder, Axelle; Sparkes, Imogen; Hawes, Chris; Popineau, Yves

2011-01-01

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP ‘core’. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions. PMID:21617248

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2.

PubMed

Vancraenenbroeck, Renée; Lobbestael, Evy; Weeks, Stephen D; Strelkov, Sergei V; Baekelandt, Veerle; Taymans, Jean-Marc; De Maeyer, Marc

2012-03-01

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions. Copyright © 2011 Elsevier B.V. All rights reserved.

Multifunctional G-Rich and RRM-Containing Domains of TbRGG2 Perform Separate yet Essential Functions in Trypanosome RNA Editing

PubMed Central

Foda, Bardees M.; Downey, Kurtis M.; Fisk, John C.

2012-01-01

Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3′-to-5′ progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain. PMID:22798390

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

PubMed Central

Burtscher, Laura; Hajdu, Dorottya; Muñoz, Alberto; Gáspári, Zoltán; Read, Nick D.; Batta, Gyula; Marx, Florentine

2017-01-01

The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF. PMID:28072824

Importance of Conserved Cysteine Residues in the Coronavirus Envelope Protein▿

PubMed Central

Lopez, Lisa A.; Riffle, Ambere J.; Pike, Steven L.; Gardner, Douglas; Hogue, Brenda G.

2008-01-01

Coronavirus envelope (E) proteins play an important, not fully understood role(s) in the virus life cycle. All E proteins have conserved cysteine residues located on the carboxy side of the long hydrophobic domain, suggesting functional significance. In this study, we confirmed that mouse hepatitis coronavirus A59 E protein is palmitoylated. To understand the role of the conserved residues and the necessity of palmitoylation, three cysteines at positions 40, 44, and 47 were changed singly and in various combinations to alanine. Double- and triple-mutant E proteins resulted in decreased virus-like particle output when coexpressed with the membrane (M) protein. Mutant E proteins were also studied in the context of a full-length infectious clone. Single-substitution viruses exhibited growth characteristics virtually identical to those of the wild-type virus, while the double-substitution mutations gave rise to viruses with less robust growth phenotypes indicated by smaller plaques and decreased virus yields. In contrast, replacement of all three cysteines resulted in crippled virus with significantly reduced yields. Triple-mutant viruses did not exhibit impairment in entry. Mutant E proteins localized properly in infected cells. A comparison of intracellular and extracellular virus yields suggested that release is only slightly impaired. E protein lacking all three cysteines exhibited an increased rate of degradation compared to that of the wild-type protein, suggesting that palmitoylation is important for the stability of the protein. Altogether, the results indicate that the conserved cysteines and presumably palmitoylation are functionally important for virus production. PMID:18184703

Convergent solid-phase and solution approaches in the synthesis of the cysteine-rich Mdm2 RING finger domain.

PubMed

Vasileiou, Zoe; Barlos, Kostas; Gatos, Dimitrios

2009-12-01

The RING finger domain of the Mdm2, located at the C-terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48-residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid-phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C-terminus as the amino component. Best results were achieved using solution condensation where the N-component was applied with the C-terminal carboxyl group left unprotected. The developed method is well suited for large-scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid-phase and solution synthesis. (c) 2009 European Peptide Society and John Wiley & Sons, Ltd.

Dipeptide frequency/bias analysis identifies conserved sites of nonrandomness shared by cysteine-rich motifs.

PubMed

Campion, S R; Ameen, A S; Lai, L; King, J M; Munzenmaier, T N

2001-08-15

This report describes the application of a simple computational tool, AAPAIR.TAB, for the systematic analysis of the cysteine-rich EGF, Sushi, and Laminin motif/sequence families at the two-amino acid level. Automated dipeptide frequency/bias analysis detects preferences in the distribution of amino acids in established protein families, by determining which "ordered dipeptides" occur most frequently in comprehensive motif-specific sequence data sets. Graphic display of the dipeptide frequency/bias data revealed family-specific preferences for certain dipeptides, but more importantly detected a shared preference for employment of the ordered dipeptides Gly-Tyr (GY) and Gly-Phe (GF) in all three protein families. The dipeptide Asn-Gly (NG) also exhibited high-frequency and bias in the EGF and Sushi motif families, whereas Asn-Thr (NT) was distinguished in the Laminin family. Evaluation of the distribution of dipeptides identified by frequency/bias analysis subsequently revealed the highly restricted localization of the G(F/Y) and N(G/T) sequence elements at two separate sites of extreme conservation in the consensus sequence of all three sequence families. The similar employment of the high-frequency/bias dipeptides in three distinct protein sequence families was further correlated with the concurrence of these shared molecular determinants at similar positions within the distinctive scaffolds of three structurally divergent, but similarly employed, motif modules.

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome.

PubMed

Faheem, Muhammad; Martins-de-Sa, Diogo; Vidal, Julia F D; Álvares, Alice C M; Brandão-Neto, José; Bird, Louise E; Tully, Mark D; von Delft, Frank; Souto, Betulia M; Quirino, Betania F; Freitas, Sonia M; Barbosa, João Alexandre R G

2016-12-09

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5  M -1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.

Modification by covalent reaction or oxidation of cysteine residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding

PubMed Central

Visperas, Patrick R.; Winger, Jonathan A.; Horton, Timothy M.; Shah, Neel H.; Aum, Diane J.; Tao, Alyssa; Barros, Tiago; Yan, Qingrong; Wilson, Christopher G.; Arkin, Michelle R.; Weiss, Arthur; Kuriyan, John

2015-01-01

Zeta-chain Associated Protein of 70kDa (ZAP-70) and Spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signaling, respectively. They are recruited, via their tandem-SH2 domains, to doubly-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signaling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys 39 in ZAP-70, Cys 206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of hydrogen peroxide, and these two cysteine residues are also necessary for inhibition by hydrogen peroxide. Our findings suggest a mechanism by which the generation of reactive oxygen species generated during responses to antigen could attenuate signaling through these kinases, and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains. PMID:25287889

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

PubMed Central

Kim, Jin Sun; Park, Su Jung; Kwak, Kyung Jin; Kim, Yeon Ok; Kim, Joo Yeol; Song, Jinkyung; Jang, Boseung; Jung, Che-Hun; Kang, Hunseung

2007-01-01

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process. PMID:17169986

High secreted protein acidic and rich in cysteine expression in peritumoral fibroblasts predicts better prognosis in patients with resectable gastric cancer

PubMed Central

Nakajima, Masao; Yoshino, Shigefumi; Kanekiyo, Shinsuke; Maeda, Noriko; Sakamoto, Kazuhiko; Tsunedomi, Ryoichi; Suzuki, Nobuaki; Takeda, Shigeru; Yamamoto, Shigeru; Hazama, Shoichi; Hoshii, Yoshinobu; Oga, Atsunori; Itoh, Hiroshi; Ueno, Tomio; Nagano, Hiroaki

2018-01-01

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix glycoprotein that may serve an important role in epithelial-mesenchymal transition. Recent studies have demonstrated that SPARC status is a prognostic indicator in various cancer types; however, its value remains unclear in gastric cancer (GC). In the present study, the localization and prognostic impact of SPARC expression were evaluated in patients with GC. Immunohistochemical analysis of SPARC expression was performed in 117 surgically resected GC specimens, and the localization of SPARC positive cells, as well as the rassociation between SPARC expression and clinicopathological characteristics were evaluated. High SPARC expression was observed in 47 cases; the glycoprotein was localized in the peritumoral fibroblasts, but was rarely observed in the cytoplasm of cancer cells. Heterogeneity of SPARC expression was observed in 52 cases. High stromal SPARC expression was identified to be an independent predictor of more favorable prognosis (overall survival and recurrence free survival) in all patients (P<0.001). On subgroup analysis, this association remained significant in patients who received adjuvant chemotherapy, but not in patients who did not (P<0.001). Stromal SPARC expression predicts better prognosis in GC patients who underwent curative resection; this appears to be associated with improved response to chemotherapy. PMID:29403557

Epididymal secreted protein Crisp-1 and sperm function.

PubMed

Roberts, Kenneth P; Ensrud, Kathy M; Wooters, Joseph L; Nolan, Michael A; Johnston, Daniel S; Hamilton, David W

2006-05-16

Crisp-1 is a member of the cysteine-rich secretory protein family. This family of proteins is characterized by the presence of 16 conserved cysteine residues, the characteristic from which the family name is derived. Members of the Crisp protein family are found in the secretions of the reproductive tract and salivary glands, including venom toxins from several species of snakes and lizards. The Crisp proteins are modular, each containing an amino terminal pathogenesis-related (PR)-like domain and a carboxyl terminal cysteine-rich domain (CRD) connected by a hinge region. Sequence and structural similarities to proteins with known functions suggest that the Crisp family of proteins may act by regulating cellular ion channels. Rat Crisp-1 is synthesized as two distinct isoforms (referred to as Proteins D and E) by the epididymal epithelium and both are secreted into the luminal fluid where they interact with spermatozoa. Our laboratory has correlated Crisp-1 binding to sperm with inhibiting the signaling cascades that initiate capacitation while others have shown that blocking Crisp-1 binding sites on oocytes interferes with sperm-egg fusion. We hypothesize that the D and E populations of rat Crisp-1 have different interactions with sperm that modulate these distinct biological activities. Through tandem mass spectrometry (MS/MS) and monosaccharide composition analyses, we have identified at least one difference between the D and E forms as an additional single O-linked N-acetyl galactosamine on an amino terminal threonine residue in Protein E. This post-translational modification appears to account for the unique 'E' epitope bound by monoclonal antibody 4E9 developed in our laboratory, and may also lead to differential processing and localization of Protein E on sperm, when compared to Protein D. These findings are the first step in distinguishing the molecular basis of the biological activities of the D and E forms of rat Crisp-1. The epididymal

Overexpression of serine acetlytransferase produced large increases in O-acetylserine and free cysteine in developing seeds of a grain legume

PubMed Central

Tabe, Linda; Wirtz, Markus; Molvig, Lisa; Droux, Michel; Hell, Ruediger

2010-01-01

There have been many attempts to increase concentrations of the nutritionally essential sulphur amino acids by modifying their biosynthetic pathway in leaves of transgenic plants. This report describes the first modification of cysteine biosyntheis in developing seeds; those of the grain legume, narrow leaf lupin (Lupinus angustifolius, L.). Expression in developing lupin embryos of a serine acetyltransferase (SAT) from Arabidopsis thaliana (AtSAT1 or AtSerat 2;1) was associated with increases of up to 5-fold in the concentrations of O-acetylserine (OAS), the immediate product of SAT, and up to 26-fold in free cysteine, resulting in some of the highest in vivo concentrations of these metabolites yet reported. Despite the dramatic changes in free cysteine in developing embryos of SAT overexpressers, concentrations of free methionine in developing embryos, and the total cysteine and methionine concentrations in mature seeds were not significantly altered. Pooled F2 seeds segregating for the SAT transgene and for a transgene encoding a methionine- and cysteine-rich sunflower seed storage protein also had increased OAS and free cysteine, but not free methionine, during development, and no increase in mature seed total sulphur amino acids compared with controls lacking SAT overexpression. The data support the view that the cysteine biosynthetic pathway is active in developing seeds, and indicate that SAT activity limits cysteine biosynthesis, but that cysteine supply is not limiting for methionine biosynthesis or for storage protein synthesis in maturing lupin embryos in conditions of adequate sulphur nutrition. OAS and free methionine, but not free cysteine, were implicated as signalling metabolites controlling expression of a gene for a cysteine-rich seed storage protein. PMID:19939888

Overexpression of serine acetlytransferase produced large increases in O-acetylserine and free cysteine in developing seeds of a grain legume.

PubMed

Tabe, Linda; Wirtz, Markus; Molvig, Lisa; Droux, Michel; Hell, Ruediger

2010-03-01

There have been many attempts to increase concentrations of the nutritionally essential sulphur amino acids by modifying their biosynthetic pathway in leaves of transgenic plants. This report describes the first modification of cysteine biosynthesis in developing seeds; those of the grain legume, narrow leaf lupin (Lupinus angustifolius, L.). Expression in developing lupin embryos of a serine acetyltransferase (SAT) from Arabidopsis thaliana (AtSAT1 or AtSerat 2;1) was associated with increases of up to 5-fold in the concentrations of O-acetylserine (OAS), the immediate product of SAT, and up to 26-fold in free cysteine, resulting in some of the highest in vivo concentrations of these metabolites yet reported. Despite the dramatic changes in free cysteine in developing embryos of SAT overexpressers, concentrations of free methionine in developing embryos, and the total cysteine and methionine concentrations in mature seeds were not significantly altered. Pooled F(2) seeds segregating for the SAT transgene and for a transgene encoding a methionine- and cysteine-rich sunflower seed storage protein also had increased OAS and free cysteine, but not free methionine, during development, and no increase in mature seed total sulphur amino acids compared with controls lacking SAT overexpression. The data support the view that the cysteine biosynthetic pathway is active in developing seeds, and indicate that SAT activity limits cysteine biosynthesis, but that cysteine supply is not limiting for methionine biosynthesis or for storage protein synthesis in maturing lupin embryos in conditions of adequate sulphur nutrition. OAS and free methionine, but not free cysteine, were implicated as signalling metabolites controlling expression of a gene for a cysteine-rich seed storage protein.

Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein.

PubMed

Hu, A; Norrby, E

1994-09-01

The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties. (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface. (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not

Inferring Domain-Domain Interactions from Protein-Protein Interactions with Formal Concept Analysis

PubMed Central

Khor, Susan

2014-01-01

Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains. PMID:24586450

Redox Signaling Regulated by Cysteine Persulfide and Protein Polysulfidation.

PubMed

Kasamatsu, Shingo; Nishimura, Akira; Morita, Masanobu; Matsunaga, Tetsuro; Abdul Hamid, Hisyam; Akaike, Takaaki

2016-12-15

For decades, reactive persulfide species including cysteine persulfide (CysSSH) have been known to exist endogenously in organisms. However, the physiological significance of endogenous persulfides remains poorly understood. That cystathionine β-synthase and cystathionine γ-lyase produced CysSSH from cystine was recently demonstrated. An endogenous sulfur transfer system involving CysSSH evidently generates glutathione persulfide (GSSH) that exists at concentrations greater than 100 μM in vivo. Because reactive persulfide species such as CysSSH and GSSH have higher nucleophilicity than parental cysteine (Cys) and glutathione do, these reactive species exhibit strong scavenging activities against oxidants, e.g., hydrogen peroxide, and electrophiles, which contributes to redox signaling regulation. Also, several papers indicated that various proteins and enzymes have Cys polysulfides including CysSSH at their specific Cys residues, which is called protein polysulfidation. Apart from the redox signaling regulatory mechanism, another plausible function of protein polysulfidation is providing protection for protein thiol residues against irreversible chemical modification caused by oxidants and electrophiles. Elucidation of the redox signaling regulatory mechanism of reactive persulfide species including small thiol molecules and thiol-containing proteins should lead to the development of new therapeutic strategies and drug discoveries for oxidative and electrophilic stress-related diseases.

Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

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

Latonen, Leena; Jaervinen, Paeivi M.; Haartman Institute, University of Helsinki, FIN-00014 Helsinki

2008-02-15

Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1more » correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions.« less

Activation of the N-Terminally Truncated Form of the Stk Receptor Tyrosine Kinase Sf-Stk by Friend Virus-Encoded gp55 Is Mediated by Cysteine Residues in the Ecotropic Domain of gp55 and the Extracellular Domain of Sf-Stk ▿

PubMed Central

He, Shihan; Ni, Shuang; Hegde, Shailaja; Wang, Xin; Sharda, Daniel R.; August, Avery; Paulson, Robert F.; Hankey, Pamela A.

2010-01-01

Friend virus induces an erythroleukemia in susceptible mice that is initiated by the interaction of the Friend virus-encoded glycoprotein gp55 with the erythropoietin (Epo) receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk, recruitment of a Grb2/Gab2/Stat3 signaling complex, and induction of Pu.1 expression by Stat3 are required for the development of the early stage of Friend disease both in vitro and in vivo. Here we demonstrate that the interaction of gp55 with Sf-Stk is dependent on cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk. Point mutation of these cysteine residues or deletion of these domains inhibits the ability of gp55 to interact with Sf-Stk, resulting in the inability of these proteins to promote the Epo-independent growth of erythroid progenitor cells. We also demonstrate that the interaction of gp55 with Sf-Stk does not promote dimerization of Sf-Stk but results in enhanced phosphorylation of Sf-Stk and the relocalization of Sf-Stk from the cytosol to the plasma membrane. Finally, we demonstrate that a constitutively active form of Sf-Stk (Sf-StkM330T), as well as its human counterpart, Sf-Ron, promotes Epo-independent colony formation in the absence of gp55 and that this response is also dependent on the cysteines in the extracellular domains of Sf-StkM330T and Sf-Ron. These data suggest that the cysteines in the extracellular domains of Sf-Stk and Sf-Ron may also mediate the interaction of these truncated receptors with other cellular factors that regulate their ability to promote cytokine-independent growth. PMID:20016000

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica

PubMed Central

Wong, Ka H.; Tan, Wei Liang; Kini, Shruthi G.; Xiao, Tianshu; Serra, Aida; Sze, Sui Kwan; Tam, James P.

2017-01-01

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40–41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics. PMID:28680440

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica.

PubMed

Wong, Ka H; Tan, Wei Liang; Kini, Shruthi G; Xiao, Tianshu; Serra, Aida; Sze, Sui Kwan; Tam, James P

2017-01-01

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40-41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC 50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics.

Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

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

Heintz, Udo; Meinhart, Anton; Winkler, Andreas, E-mail: andreas.winkler@mpimf-heidelberg.mpg.de

2014-03-01

Crystal structures of two truncated variants of the transcription factor PpsR from R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function. Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteinsmore » are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsR{sub Q-PAS1}) and two (PpsR{sub N-Q-PAS1}) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR{sub ΔHTH}) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR{sub ΔHTH} structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N-domain

Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

PubMed

Rutten, Julie W; Dauwerse, Hans G; Peters, Dorien J M; Goldfarb, Andrew; Venselaar, Hanka; Haffner, Christof; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke M; Lesnik Oberstein, Saskia A J

2016-04-01

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in the NOTCH3 gene. NOTCH3 mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specific NOTCH3 exons. Selection of these exons was achieved using in silico studies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targeted NOTCH3 exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation*

PubMed Central

Ho, Tina; Watt, Brenda; Spruce, Lynn A.; Seeholzer, Steven H.; Marks, Michael S.

2016-01-01

The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils. PMID:26694611

SPARC (secreted protein acidic and rich in cysteine) knockdown protects mice from acute liver injury by reducing vascular endothelial cell damage

PubMed Central

Peixoto, E; Atorrasagasti, C; Aquino, JB; Militello, R; Bayo, J; Fiore, E; Piccioni, F; Salvatierra, E; Alaniz, L; García, MG; Bataller, R; Corrales, F; Gidekel, M; Podhajcer, O; Colombo, MI; Mazzolini, G

2015-01-01

Secreted protein, acidic and rich in cysteine (SPARC) is involved in many biological process including liver fibrogenesis, but its role in acute liver damage is unknown. To examine the role of SPARC in acute liver injury, we used SPARC knock-out (SPARC−/−) mice. Two models of acute liver damage were used: concanavalin A (Con A) and the agonistic anti-CD95 antibody Jo2. SPARC expression levels were analyzed in liver samples from patients with acute-on-chronic alcoholic hepatitis (AH). SPARC expression is increased on acute-on-chronic AH patients. Knockdown of SPARC decreased hepatic damage in the two models of liver injury. SPARC−/− mice showed a marked reduction in Con A-induced necroinflammation. Infiltration by CD4+ T cells, expression of tumor necrosis factor-α and interleukin-6 and apoptosis were attenuated in SPARC−/− mice. Sinusoidal endothelial cell monolayer was preserved and was less activated in Con A-treated SPARC−/− mice. SPARC knockdown reduced Con A-induced autophagy of cultured human microvascular endothelial cells (HMEC-1). Hepatic transcriptome analysis revealed several gene networks that may have a role in the attenuated liver damaged found in Con A-treated SPARC−/− mice. SPARC has a significant role in the development of Con A-induced severe liver injury. These results suggest that SPARC could represent a therapeutic target in acute liver injury. PMID:25410742

Protein domain analysis of genomic sequence data reveals regulation of LRR related domains in plant transpiration in Ficus.

PubMed

Lang, Tiange; Yin, Kangquan; Liu, Jinyu; Cao, Kunfang; Cannon, Charles H; Du, Fang K

2014-01-01

Predicting protein domains is essential for understanding a protein's function at the molecular level. However, up till now, there has been no direct and straightforward method for predicting protein domains in species without a reference genome sequence. In this study, we developed a functionality with a set of programs that can predict protein domains directly from genomic sequence data without a reference genome. Using whole genome sequence data, the programming functionality mainly comprised DNA assembly in combination with next-generation sequencing (NGS) assembly methods and traditional methods, peptide prediction and protein domain prediction. The proposed new functionality avoids problems associated with de novo assembly due to micro reads and small single repeats. Furthermore, we applied our functionality for the prediction of leucine rich repeat (LRR) domains in four species of Ficus with no reference genome, based on NGS genomic data. We found that the LRRNT_2 and LRR_8 domains are related to plant transpiration efficiency, as indicated by the stomata index, in the four species of Ficus. The programming functionality established in this study provides new insights for protein domain prediction, which is particularly timely in the current age of NGS data expansion.

Aged garlic extract and S-allyl cysteine prevent formation of advanced glycation endproducts.

PubMed

Ahmad, Muhammad Saeed; Pischetsrieder, Monika; Ahmed, Nessar

2007-04-30

Hyperglycaemia causes increased protein glycation and the formation of advanced glycation endproducts which underlie the complications of diabetes and ageing. Glycation is accompanied by metal-catalysed oxidation of glucose and Amadori products to form free radicals capable of protein fragmentation. Aged garlic extract is a potent antioxidant with established lipid-lowering effects attributed largely to a key ingredient called S-allyl cysteine. This study investigated the ability of aged garlic extract and S-allyl cysteine to inhibit advanced glycation in vitro. Bovine serum albumin (BSA) was glycated in the presence of Cu(2+) ions and different concentrations of aged garlic extract and protein fragmentation was examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Lysozyme was glycated by glucose or methylglyoxal in the presence of different concentrations of aged garlic extract or S-allyl cysteine with subsequent analysis of glycation-derived crosslinking using SDS-PAGE. Amadori-rich protein was prepared by dialysing lysozyme that had been glycated by ribose for 24 h. This ribated lysozyme was reincubated and the effects of aged garlic extract, S-allyl cysteine and pyridoxamine on glycation-induced crosslinking was monitored. Aged garlic extract inhibited metal-catalysed protein fragmentation. Both aged garlic extract and S-allyl cysteine inhibited formation of glucose and methylglyoxal derived advanced glycation endproducts and showed potent Amadorin activity when compared to pyridoxamine. S-allyl cysteine inhibited formation of carboxymethyllysine (CML), a non-crosslinked advanced glycation endproduct derived from oxidative processes. Further studies are required to assess whether aged garlic extract and S-allyl cysteine can protect against the harmful effects of glycation and free radicals in diabetes and ageing.

Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy.

PubMed

Horne, A W; Duncan, W C; King, A E; Burgess, S; Lourenco, P C; Cornes, P; Ghazal, P; Williams, A R; Udby, L; Critchley, H O D

2009-05-01

Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT-PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic

The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

PubMed Central

Frisardi, Marta; Ghosh, Sudip K.; Field, Jessica; Van Dellen, Katrina; Rogers, Rick; Robbins, Phillips; Samuelson, John

2000-01-01

The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study an extraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional protein gels of cyst walls purified from Entamoeba invadens. Jacob, which was acidic and had an apparent molecular mass of ∼100 kDa, contained sugars that bound to concanavalin A and ricin. The jacob gene encoded a 45-kDa protein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but not homologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins that surround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecyl sulfate-treated cyst walls. Conversely, the E. histolytica plasma membrane Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob. In the presence of galactose, E. invadens formed wall-less cysts, which were quadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles. A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria and mucin-coated beads. These results suggest that the E. invadens cyst wall forms when the plasma membrane galactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains. PMID:10858239

Fine mapping of a dominantly inherited powdery mildew resistance major-effect QTL, Pm1.1, in cucumber identifies a 41.1 kb region containing two tandemly arrayed cysteine-rich receptor-like protein kinase genes.

PubMed

Xu, Xuewen; Yu, Ting; Xu, Ruixue; Shi, Yang; Lin, Xiaojian; Xu, Qiang; Qi, Xiaohua; Weng, Yiqun; Chen, Xuehao

2016-03-01

A dominantly inherited major-effect QTL for powdery mildew resistance in cucumber was fine mapped. Two tandemly arrayed cysteine-rich receptor-like protein kinase genes were identified as the most possible candidates. Powdery mildew (PM) is one of the most severe fungal diseases of cucumber (Cucumis sativus L.) and other cucurbit crops, but the molecular genetic mechanisms of powdery mildew resistance in cucurbits are still poorly understood. In this study, through marker-assisted backcrossing with an elite cucumber inbred line, D8 (PM susceptible), we developed a single-segment substitution line, SSSL0.7, carrying 95 kb fragment from PM resistance donor, Jin5-508, that was defined by two microsatellite markers, SSR16472 and SSR16881. A segregating population with 3600 F2 plants was developed from the SSSL0.7 × D8 mating; segregation analysis confirmed a dominantly inherited major-effect QTL, Pm1.1 in cucumber chromosome 1 underlying PM resistance in SSSL0.7. New molecular markers were developed through exploring the next generation resequenced genomes of Jin5-508 and D8. Linkage analysis and QTL mapping in a subset of the F2 plants delimited the Pm1.1 locus into a 41.1 kb region, in which eight genes were predicted. Comparative gene expression analysis revealed that two concatenated genes, Csa1M064780 and Csa1M064790 encoding the same function of a cysteine-rich receptor-like protein kinase, were the most likely candidate genes. GFP fusion protein-aided subcellular localization indicated that both candidate genes were located in the plasma membrane, but Csa1M064780 was also found in the nucleus. This is the first report of dominantly inherited PM resistance in cucumber. Results of this study will provide new insights into understanding the phenotypic and genetic mechanisms of PM resistance in cucumber. This work should also facilitate marker-assisted selection in cucumber breeding for PM resistance.

Detecting Coevolution in and among Protein Domains

PubMed Central

Yeang, Chen-Hsiang; Haussler, David

2007-01-01

Correlated changes of nucleic or amino acids have provided strong information about the structures and interactions of molecules. Despite the rich literature in coevolutionary sequence analysis, previous methods often have to trade off between generality, simplicity, phylogenetic information, and specific knowledge about interactions. Furthermore, despite the evidence of coevolution in selected protein families, a comprehensive screening of coevolution among all protein domains is still lacking. We propose an augmented continuous-time Markov process model for sequence coevolution. The model can handle different types of interactions, incorporate phylogenetic information and sequence substitution, has only one extra free parameter, and requires no knowledge about interaction rules. We employ this model to large-scale screenings on the entire protein domain database (Pfam). Strikingly, with 0.1 trillion tests executed, the majority of the inferred coevolving protein domains are functionally related, and the coevolving amino acid residues are spatially coupled. Moreover, many of the coevolving positions are located at functionally important sites of proteins/protein complexes, such as the subunit linkers of superoxide dismutase, the tRNA binding sites of ribosomes, the DNA binding region of RNA polymerase, and the active and ligand binding sites of various enzymes. The results suggest sequence coevolution manifests structural and functional constraints of proteins. The intricate relations between sequence coevolution and various selective constraints are worth pursuing at a deeper level. PMID:17983264

Bacterial Cysteine-Inducible Cysteine Resistance Systems

PubMed Central

Takumi, Kazuhiro

2016-01-01

ABSTRACT Cysteine donates sulfur to macromolecules and occurs naturally in many proteins. Because low concentrations of cysteine are cytotoxic, its intracellular concentration is stringently controlled. In bacteria, cysteine biosynthesis is regulated by feedback inhibition of the activities of serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrogenase (3-PGDH) and is also regulated at the transcriptional level by inducing the cysteine regulon using the master regulator CysB. Here, we describe two novel cysteine-inducible systems that regulate the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for biotechnological, medical, and industrial purposes. One locus, designated ccdA (formerly PAJ_0331), encodes a novel cysteine-inducible cysteine desulfhydrase (CD) that degrades cysteine, and its expression is controlled by the transcriptional regulator encoded by ccdR (formerly PAJ_0332 or ybaO), located just upstream of ccdA. The other locus, designated cefA (formerly PAJ_3026), encodes a novel cysteine-inducible cysteine efflux pump that is controlled by the transcriptional regulator cefR (formerly PAJ_3027), located just upstream of cefA. To our knowledge, this is the first example where the expression of CD and an efflux pump is regulated in response to cysteine and is directly involved in imparting resistance to excess levels of cysteine. We propose that ccdA and cefA function as safety valves that maintain homeostasis when the intra- or extracellular cysteine concentration fluctuates. Our findings contribute important insights into optimizing the production of cysteine and related biomaterials by P. ananatis. IMPORTANCE Because of its toxicity, the bacterial intracellular cysteine level is stringently regulated at biosynthesis. This work describes the identification and characterization of two novel cysteine-inducible systems that regulate, through degradation and

Differential Evolution and Neofunctionalization of Snake Venom Metalloprotease Domains*

PubMed Central

Brust, Andreas; Sunagar, Kartik; Undheim, Eivind A.B.; Vetter, Irina; Yang, Daryl C.; Casewell, Nicholas R.; Jackson, Timothy N. W.; Koludarov, Ivan; Alewood, Paul F.; Hodgson, Wayne C.; Lewis, Richard J.; King, Glenn F.; Antunes, Agostinho; Hendrikx, Iwan; Fry, Bryan G.

2013-01-01

Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands. PMID:23242553

Differential evolution and neofunctionalization of snake venom metalloprotease domains.

PubMed

Brust, Andreas; Sunagar, Kartik; Undheim, Eivind A B; Vetter, Irina; Yang, Daryl C; Yang, Dary C; Casewell, Nicholas R; Jackson, Timothy N W; Koludarov, Ivan; Alewood, Paul F; Hodgson, Wayne C; Lewis, Richard J; King, Glenn F; Antunes, Agostinho; Hendrikx, Iwan; Fry, Bryan G

2013-03-01

Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands.

Resistance to mitomycin C requires direct interaction between the Fanconi anemia proteins FANCA and FANCG in the nucleus through an arginine-rich domain.

PubMed

Kruyt, F A; Abou-Zahr, F; Mok, H; Youssoufian, H

1999-11-26

Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, birth defects, and chromosomal instability. Because FA cells are sensitive to mitomycin C (MMC), FA gene products could be involved in cellular defense mechanisms. The FANCA and FANCG proteins deficient in FA groups A and G interact directly with each other. We have localized the mutual interaction domains of these proteins to amino acids 18-29 of FANCA and to two noncontiguous carboxyl-terminal domains of FANCG encompassing amino acids 400-475 and 585-622. Site-directed mutagenesis of FANCA residues 18-29 revealed a novel arginine-rich interaction domain (RRRAWAELLAG). By alanine mutagenesis, Arg(1), Arg(2), and Leu(8) but not Arg(3), Trp(5), and Glu(7) appeared to be critical for binding to FANCG. Similar immunolocalization for FANCA and FANCG suggested that these proteins interact in vivo. Moreover, targeting of FANCA to the nucleus or the cytoplasm with nuclear localization and nuclear export signals, respectively, showed concordance between the localization patterns of FANCA and FANCG. The complementation function of FANCA was abolished by mutations in its FANCG-binding domain. Conversely, stable expression of FANCA mutants encoding intact FANCG interaction domains induced hypersensitivity to MMC in HeLa cells. These results demonstrate that FANCA-FANCG complexes are required for cellular resistance to MMC. Because the FANCC protein deficient in FA group C works within the cytoplasm, we suggest that FANCC and the FANCA-FANCG complexes suppress MMC cytotoxicity within distinct cellular compartments.

The Arabidopsis thaliana cysteine-rich receptor-like kinase CRK20 modulates host responses to Pseudomonas syringae pv. tomato DC3000 infection.

PubMed

Ederli, Luisa; Madeo, Laura; Calderini, Ornella; Gehring, Chris; Moretti, Chiaraluce; Buonaurio, Roberto; Paolocci, Francesco; Pasqualini, Stefania

2011-10-15

In plants, the cysteine-rich repeat kinases (CRKs) are a sub-family of receptor-like protein kinases that contain the DUF26 motif in their extracellular domains. It has been shown that in Arabidopsis thaliana, CRK20 is transcriptionally induced by pathogens, salicylic acid and ozone (O(3)). However, its role in responses to biotic and abiotic stress remains to be elucidated. To determine the function of CRK20 in such responses, two CRK20 loss-of-function mutants, crk20-1 and crk20-2, were isolated from public collections of Arabidopsis T-DNA tagged lines and examined for responses to O(3) and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O(3) sensitivities and no differences in the expression of defense genes when compared with the wild-type. However, pathogen growth was significantly reduced, while there were no differences in the induction of salicylic acid related defense genes or salicylic acid accumulation. Furthermore, correlation analysis of CRK20 gene expression suggests that it has a role in the control of H(2)O and/or nutrient transport. We therefore propose that CRK20 promotes conditions that are favorable for Pst DC3000 growth in Arabidopsis, possibly through the regulation of apoplastic homeostasis, and consequently, of the environment of this biotrophic pathogen. Copyright © 2011 Elsevier GmbH. All rights reserved.

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER)

PubMed Central

Munfus, Delicia L; Haga, Christopher L; Burrows, Peter D; Cooper, Max D

2007-01-01

Background In mouse the cytokine interleukin-7 (IL-7) is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER). The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR), a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules. PMID:17854505

Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

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

Lupardus, P.J.; Shen, A.; Bogyo, M.

2009-05-19

Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

PubMed

Inglis, Steven R; Stojkoski, Cvetan; Branson, Kim M; Cawthray, Jacquie F; Fritz, Daniel; Wiadrowski, Emma; Pyke, Simon M; Booker, Grant W

2004-10-21

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

Peptidomic Identification of Cysteine-Rich Peptides from Plants.

PubMed

Hemu, Xinya; Serra, Aida; Darwis, Dina A; Cornvik, Tobias; Sze, Siu Kwan; Tam, James P

2018-01-01

Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database.

Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes.

PubMed

Suzuki, Akiko; Endo, Takeshi

2002-02-06

We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.

Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function

USDA-ARS?s Scientific Manuscript database

This study is focused on the characterization and expression of genes in the red flour beetle, Tribolium castaneum, encoding proteins that possess six-cysteine-containing chitin-binding domains (CBDs) related to the peritrophin A domain (ChtBD2). An exhaustive bioinformatics search of the genome of...

Functional domains of the Drosophila Engrailed protein.

PubMed Central

Han, K; Manley, J L

1993-01-01

We have studied the transcriptional activity of the Drosophila homeodomain protein Engrailed (En) by using a transient expression assay employing Schneider L2 cells. En was found to very strongly repress promoters activated by a variety of different activator proteins. However, unlike another Drosophila homeodomain-containing repressor, Even-skipped (Eve), En was unable to repress the activity of several basal promoters in the absence of activator expression. These findings indicate that En is a specific repressor of activated transcription, and suggest that En may repress transcription by a different mechanism than Eve, perhaps by interfering with interactions between transcriptional activators and the general transcription machinery. By analyzing the properties of a variety of En mutants, we identified a minimal repression domain composed of 55 residues, which can function when fused to a heterologous DNA binding domain. Like repression domains identified in the Drosophila repressors Eve and Krüppel, the En repression domain is rich in alanine residues (26%), but unlike these other domains, is moderately charged (six arginine and three glutamic acid residues). Separate regions of En that may in some circumstances function in transcriptional activation were also identified. Images PMID:8334991

Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

PubMed Central

Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K.; Miyazaki, Hiroshi

2014-01-01

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells. PMID:24711565

Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

PubMed

Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

2014-06-01

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

PubMed

Kroj, Thomas; Chanclud, Emilie; Michel-Romiti, Corinne; Grand, Xavier; Morel, Jean-Benoit

2016-04-01

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Cysteine-Zn2+ complexes: unique molecular switches for inducible nitric oxide synthase-derived NO.

PubMed

Kröncke, K D

2001-11-01

Nitric oxide (NO) in the low nanomolar range acts as a transcellular messenger molecule to initiate regulatory and physiological responses in nearby target cells via binding to the soluble guanylate cyclase heme moiety. Higher NO concentrations, as synthesized by the inducible NO synthase (iNOS) during inflammatory processes, show additional effects: NO may react with O2, yielding nitrogen oxides like N2O3 that are able to nitrosate thiols. A variety of proteins involved in very different functions of the cell contain cysteine-Zn2+ complexes. Effects of NO on different proteins containing cysteine-Zn2+ domains and playing essential roles during transcription, protein folding, and proteolysis are discussed. It is suggested that iNOS-derived NO acts as a signal molecule targeting cysteine-Zn2+ linkages, thus enabling cells to react toward nitrosative stress.

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

PubMed

Draeger, Christian; Ndinyanka Fabrice, Tohnyui; Gineau, Emilie; Mouille, Grégory; Kuhn, Benjamin M; Moller, Isabel; Abdou, Marie-Therese; Frey, Beat; Pauly, Markus; Bacic, Antony; Ringli, Christoph

2015-06-24

Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica.

PubMed

Zhong, Jian; Wang, Wenhong; Yang, Xiaomei; Yan, Xiuwen; Liu, Rui

2013-01-01

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study.

PubMed

Cao, Rui; Wang, T Tiffany; DeMaria, Genevieve; Sheehan, John K; Kesimer, Mehmet

2012-08-03

Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non

Nucleotide-Binding Domain Leucine-Rich Repeat Containing Proteins and Intestinal Microbiota: Pivotal Players in Colitis and Colitis-Associated Cancer Development.

PubMed

Prossomariti, Anna; Sokol, Harry; Ricciardiello, Luigi

2018-01-01

The nucleotide-binding domain leucine-rich repeat containing (NLR) proteins play a fundamental role in innate immunity and intestinal tissue repair. A dysbiotic intestinal microbiota, developed as a consequence of alterations in NLR proteins, has recently emerged as a crucial hit for the development of ulcerative colitis (UC) and colitis-associated cancer (CAC). The concept of the existence of functional axes interconnecting bacteria with NLR proteins in a causal role in intestinal inflammation and CAC aroused a great interest for the potential development of preventive and therapeutic strategies against UC and CAC. However, the most recent scientific evidence, which highlights many confounding factors in studies based on microbiota characterization, underlines the need for an in-depth reconsideration of the data obtained until now. The purpose of this review is to discuss the recent findings concerning the cross talk between the NLR signaling and the intestinal microbiota in UC and CAC development, and to highlight the open issues that should be explored and addressed in future studies.

Expression and localization of cysteine-rich secretory protein-3 (CRISP-3) in the prepubertal and postpubertal male horse.

PubMed

Fedorka, C E; Scoggin, K E; Squires, E L; Ball, B A; Troedsson, M H T

2017-01-01

The seminal plasma protein, cysteine-rich secretory protein-3 (CRISP-3), has been correlated with increased fertility and first-cycle conception rates, and has been suggested to be involved in the modulation of polymorphonuclear neutrophil and phagocytosis of spermatozoa during the inflammatory response to breeding in the horse. Previous research demonstrated that equine CRISP-3 is located in both the ampulla of the vas deferens and the seminal vesicles. However, this was done with nonquantitative laboratory techniques. In humans and rodents, CRISP-3 has been described as an androgen-dependent protein, but the effect of androgens on the expression of CRISP-3 has not been investigated in the horse. The objectives of this study were to (a) confirm and quantify the expression of CRISP-3 in the male equine reproductive tract, (b) describe the localization of CRISP-3 within the specific tissues which express it, and (c) determine if expression of CRISP-3 increases after puberty. We hypothesized that expression of CRISP-3 would be expressed in both the ampulla of the vas deferens and the seminal vesicles, and expression would increase after puberty. Tissues were collected postmortem from three prepubertal colts (<6 months) and six postpubertal stallions (>3 years). Tissue samples were collected from the ampulla of vas deferens, seminal vesicles, bulbourethral gland, prostate gland, testis, as well as the cauda, corpus, and caput aspects of the epididymis. Quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) were performed using an equine-specific CRISP-3 designed primer and monocolonal antibody. A mixed linear additive model was used to compare mRNA expression between age groups, and significance was set to P < 0.05. There was a significant interaction between maturity and tissue type (P < 0.0001). Expression of CRISP-3 mRNA was found primarily in the ampulla of vas deferens with lesser expression in the seminal vesicles. Expression of

Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains.

PubMed

Domeradzka, Natalia E; Werten, Marc W T; de Vries, Renko; de Wolf, Frits A

2016-06-10

Specific coupling of de novo designed recombinant protein polymers for the construction of precisely structured nanomaterials is of interest for applications in biomedicine, pharmaceutics and diagnostics. An attractive coupling strategy is to incorporate specifically interacting peptides into the genetic design of the protein polymers. An example of such interaction is the binding of particular proline-rich ligands by so-called WW-domains. In this study, we investigated whether these domains can be produced in the yeast Pichia pastoris as part of otherwise non-interacting protein polymers, and whether they bring about polymer coupling upon mixing. We constructed two variants of a highly hydrophilic protein-based polymer that differ only in their C-terminal extensions. One carries a C-terminal WW domain, and the other a C-terminal proline-rich ligand (PPxY). Both polymers were produced in P. pastoris with a purified protein yield of more than 2 g L(-1) of cell-free broth. The proline-rich module was found to be O-glycosylated, and uncommonly a large portion of the attached oligosaccharides was phosphorylated. Glycosylation was overcome by introducing a Ser → Ala mutation in the PPxY peptide. Tryptophan fluorescence monitored during titration of the polymer containing the WW domain with either the glycosylated or nonglycosylated PPxY-containing polymer revealed binding. The complementary polymers associated with a Kd of ~3 µM, regardless of glycosylation state of the PPxY domain. Binding was confirmed by isothermal titration calorimetry, with a Kd of ~9 µM. This article presents a blueprint for the production in P. pastoris of protein polymers that can be coupled using the noncovalent interaction between WW domains and proline-rich ligands. The availability of this highly specific coupling tool will hereafter allow us to construct various supramolecular structures and biomaterials.

Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels.

PubMed Central

Harrington, Melissa A; Kopito, Ron R

2002-01-01

Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATPgammaS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance. PMID:11867445

Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato

PubMed Central

Shcherbakova, Larisa A.; Odintsova, Tatyana I.; Stakheev, Alexander A.; Fravel, Deborah R.; Zavriev, Sergey K.

2016-01-01

The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed. PMID:26779237

Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane: methodology development and its application to studies of influenza viral membrane.

PubMed Central

Kawasaki, K; Yin, J J; Subczynski, W K; Hyde, J S; Kusumi, A

2001-01-01

A pulse saturation-recovery electron paramagnetic resonance (EPR) method has been developed that allows estimation of the exchange rates of a spin-labeled lipid between the bulk domain and the protein-rich membrane domain, in which the rate of collision between the spin label and molecular oxygen is reduced (slow-oxygen transport domain, or SLOT domain). It is based on the measurements of saturation-recovery signals of a lipid spin label as a function of concentrations of both molecular oxygen and the spin label. Influenza viral membrane, one of the simplest paradigms for the study of biomembranes, showed the presence of two membrane domains with slow and fast collision rates with oxygen (a 16-fold difference) at 30 degrees C. The outbound rate from and the inbound rate into the SLOT domain (or possibly the rate of the domain disintegration and formation) were estimated to be 7.7 x 10(4) and 4.6 x 10(4) s(-1), (15 micros residency time), respectively, indicating that the SLOT domain is highly dynamic and that the entire SLOT domain represents about one-third of the membrane area. Because the oxygen transport rate in the SLOT domain is a factor of two smaller than that in purple membrane, where bacteriorhodopsin is aggregated, we propose that the SLOT domain in the viral membrane is the cholesterol-rich raft domain stabilized by the trimers of hemagglutinin and/or the tetramers of neuraminidase. PMID:11159441

Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

NASA Astrophysics Data System (ADS)

Zhang, Xianjun; Zhao, Fei; Wu, Yiran; Yang, Jun; Han, Gye Won; Zhao, Suwen; Ishchenko, Andrii; Ye, Lintao; Lin, Xi; Ding, Kang; Dharmarajan, Venkatasubramanian; Griffin, Patrick R.; Gati, Cornelius; Nelson, Garrett; Hunter, Mark S.; Hanson, Michael A.; Cherezov, Vadim; Stevens, Raymond C.; Tan, Wenfu; Tao, Houchao; Xu, Fei

2017-05-01

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

NASA Astrophysics Data System (ADS)

Ogura, Kenji; Okamura, Hideyasu

2013-10-01

Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3.

The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain.

PubMed

Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J; Sivakumaran, Andrew; Yoon, Je-Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, B Göran; Wilce, Jacqueline A; Díaz-Moreno, Irene

2014-01-01

T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.

Identifying different transcribed proteins in the newly described Theraphosidae Pamphobeteus verdolaga.

PubMed

Estrada-Gómez, Sebastian; Vargas-Muñoz, Leidy Johana; Saldarriaga-Córdoba, Mónica; Cifuentes, Yeimy; Perafan, Carlos

2017-04-01

Theraphosidae spider venoms are well known for possess a complex mixture of protein and non-protein compounds in their venom. The objective of this study was to report and identify different proteins translated from the venom gland DNA information of the recently described Theraphosidae spider Pamphobeteus verdolaga. Using a venom gland transcriptomic analysis, we reported a set of the first complete sequences of seven different proteins of the recenlty described Theraphosidae spider P. verdolaga. Protein analysis indicates the presence of different proteins on the venom composition of this new spider, some of them uncommon in the Theraphosidae family. MS/MS analysis of P. verdolaga showed different fragments matching sphingomyelinases (sicaritoxin), barytoxins, hexatoxins, latroinsectotoxins, and linear (zadotoxins) peptides. Only four of the MS/MS fragments showed 100% sequence similarity with one of the transcribed proteins. Transcriptomic analysis showed the presence of different groups of proteins like phospholipases, hyaluronidases, inhibitory cysteine knots (ICK) peptides among others. The three database of protein domains used in this study (Pfam, SMART and CDD) showed congruency in the search of unique conserved protein domain for only four of the translated proteins. Those proteins matched with EF-hand proteins, cysteine rich secretory proteins, jingzhaotoxins, theraphotoxins and hexatoxins, from different Mygalomorphae spiders belonging to the families Theraphosidae, Barychelidae and Hexathelidae. None of the analyzed sequences showed a complete 100% similarity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

PubMed

Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab

2016-12-01

Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a K m of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

[Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States].

PubMed

Melnik, T N; Nagibina, G S; Surin, A K; Glukhova, K A; Melnik, B S

2018-01-01

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

Efficient synthesis of cysteine-rich cyclic peptides through intramolecular native chemical ligation of N-Hnb-Cys peptide crypto-thioesters.

PubMed

Terrier, Victor P; Delmas, Agnès F; Aucagne, Vincent

2017-01-04

We herein introduce a straightforward synthetic route to cysteine-containing cyclic peptides based on the intramolecular native chemical ligation of in situ generated thioesters. Key precursors are N-Hnb-Cys crypto-thioesters, easily synthesized by Fmoc-based SPPS. The strategy is applied to a representative range of naturally occurring cyclic disulfide-rich peptide sequences.

The s48/45 six-cysteine proteins: mediators of interaction throughout the Plasmodium life cycle.

PubMed

Arredondo, Silvia A; Kappe, Stefan H I

2017-06-01

During their life cycle Plasmodium parasites rely upon an arsenal of proteins that establish key interactions with the host and vector, and between the parasite sexual stages, with the purpose of ensuring infection, reproduction and proliferation. Among these is a group of secreted or membrane-anchored proteins known as the six-cysteine (6-cys) family. This is a small but important family with only 14 members thus far identified, each stage-specifically expressed during the parasite life cycle. 6-cys proteins often localise at the parasite surface or interface with the host and vector, and are conserved in different Plasmodium species. The unifying feature of the family is the s48/45 domain, presumably involved in adhesion and structurally related to Ephrins, the ligands of Eph receptors. The most prominent s48/45 members are currently under functional investigation and are being pursued as vaccine candidates. In this review, we examine what is known about the 6-cys family, their structure and function, and discuss future research directions. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

A cysteine-rich plant protein potentiates Potyvirus movement through an interaction with the virus genome-linked protein VPg.

PubMed

Dunoyer, P; Thomas, C; Harrison, S; Revers, F; Maule, A

2004-03-01

We have identified a cellular factor that interacts with the virus genome-linked proteins (VPgs) of a diverse range of potyviruses. The factor, called Potyvirus VPg-interacting protein (PVIP), is a plant-specific protein with homologues in all the species examined, i.e., pea, Arabidopsis thaliana, and Nicotiana benthamiana. The sequence of PVIP does not identify a specific function, although the existence of a "PHD finger" domain may implicate the protein in transcriptional control through chromatin remodeling. Deletion analysis using the yeast two-hybrid system showed that the determinants of the interaction lay close to the N terminus of VPg; indeed, the N-terminal 16 amino acids were shown to be both necessary and sufficient for the interaction with at least one PVIP protein. From a sequence comparison of different potyvirus VPg proteins, a specific amino acid at position 12 was directly implicated in the interaction. This part of VPg is distinct from regions associated with other functional roles of VPg. Through mutation of Turnip mosaic virus (TuMV) at VPg position 12, we showed that the interaction with PVIP affected systemic symptoms in infected plants. This resulted from reduced cell-to-cell and systemic movement more than reduced virus replication, as visualized by comparing green fluorescent protein-tagged wild-type and mutant viruses. Furthermore, by using RNA interference of PVIP in Arabidopsis, we showed that reduced expression of PVIP genes reduced susceptibility to TuMV infection. We conclude that PVIP functions as an ancillary factor to support potyvirus movement in plants.

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

PubMed Central

Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

2016-01-01

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

PubMed Central

Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P.

2016-01-01

Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

Apolipoprotein A-I mutant proteins having cysteine substitutions and polynucleotides encoding same

DOEpatents

Oda, Michael N [Benicia, CA; Forte, Trudy M [Berkeley, CA

2007-05-29

Functional Apolipoprotein A-I mutant proteins, having one or more cysteine substitutions and polynucleotides encoding same, can be used to modulate paraoxonase's arylesterase activity. These ApoA-I mutant proteins can be used as therapeutic agents to combat cardiovascular disease, atherosclerosis, acute phase response and other inflammatory related diseases. The invention also includes modifications and optimizations of the ApoA-I nucleotide sequence for purposes of increasing protein expression and optimization.

Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster

PubMed Central

Menger, Katja E.; James, Andrew M.; Cochemé, Helena M.; Harbour, Michael E.; Chouchani, Edward T.; Ding, Shujing; Fearnley, Ian M.; Partridge, Linda; Murphy, Michael P.

2015-01-01

Summary Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster. PMID:26095360

Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri.

PubMed

Martínez-Castillo, Moisés; Ramírez-Rico, Gerardo; Serrano-Luna, Jesús; Shibayama, Mineko

2015-01-01

Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.

Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri

PubMed Central

Ramírez-Rico, Gerardo; Serrano-Luna, Jesús; Shibayama, Mineko

2015-01-01

Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system. PMID:26090408

The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

PubMed

Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

2009-02-01

In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

Supra-domains: evolutionary units larger than single protein domains.

PubMed

Vogel, Christine; Berzuini, Carlo; Bashton, Matthew; Gough, Julian; Teichmann, Sarah A

2004-02-20

Domains are the evolutionary units that comprise proteins, and most proteins are built from more than one domain. Domains can be shuffled by recombination to create proteins with new arrangements of domains. Using structural domain assignments, we examined the combinations of domains in the proteins of 131 completely sequenced organisms. We found two-domain and three-domain combinations that recur in different protein contexts with different partner domains. The domains within these combinations have a particular functional and spatial relationship. These units are larger than individual domains and we term them "supra-domains". Amongst the supra-domains, we identified some 1400 (1203 two-domain and 166 three-domain) combinations that are statistically significantly over-represented relative to the occurrence and versatility of the individual component domains. Over one-third of all structurally assigned multi-domain proteins contain these over-represented supra-domains. This means that investigation of the structural and functional relationships of the domains forming these popular combinations would be particularly useful for an understanding of multi-domain protein function and evolution as well as for genome annotation. These and other supra-domains were analysed for their versatility, duplication, their distribution across the three kingdoms of life and their functional classes. By examining the three-dimensional structures of several examples of supra-domains in different biological processes, we identify two basic types of spatial relationships between the component domains: the combined function of the two domains is such that either the geometry of the two domains is crucial and there is a tight constraint on the interface, or the precise orientation of the domains is less important and they are spatially separate. Frequently, the role of the supra-domain becomes clear only once the three-dimensional structure is known. Since this is the case for only a

Recovering Protein-Protein and Domain-Domain Interactions from Aggregation of IP-MS Proteomics of Coregulator Complexes

PubMed Central

Mazloom, Amin R.; Dannenfelser, Ruth; Clark, Neil R.; Grigoryan, Arsen V.; Linder, Kathryn M.; Cardozo, Timothy J.; Bond, Julia C.; Boran, Aislyn D. W.; Iyengar, Ravi; Malovannaya, Anna; Lanz, Rainer B.; Ma'ayan, Avi

2011-01-01

Coregulator proteins (CoRegs) are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP) followed by mass spectrometry (MS) applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/. PMID:22219718

Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

DOE PAGES

Zhang, Xianjun; Zhao, Fei; Wu, Yiran; ...

2017-05-17

Here, the Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combiningmore » the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.« less

Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.

PubMed

Khan, Samir A; Rossi, Ana M; Riley, Andrew M; Potter, Barry V L; Taylor, Colin W

2013-04-15

IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3. Activation of IP(3)R is initiated by IP(3) binding to the IBC (IP(3)-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 μM) stimulated IP(3) binding to the isolated NT (N-terminal; residues 1-604) of IP(3)R1 and IP(3)R2, but not to that of IP(3)R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP(3)) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP(3)R activation. IP(3) binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP(3)R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP(3) binding to the chimaeric NT and IP(3)-evoked Ca(2+) release from the chimaeric IP(3)R. This is the first systematic analysis of the effects of a thiol reagent on each IP(3)R subtype. We conclude that thimerosal selectively sensitizes IP(3)R1 and IP(3)R2 to IP(3) by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

Subtype-selective regulation of IP3 receptors by thimerosal via cysteine residues within the IP3-binding core and suppressor domain

PubMed Central

Khan, Samir A.; Rossi, Ana M.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

2013-01-01

IP3R (IP3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP3R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP3-evoked Ca2+ release via IP3R1 and IP3R2, but inhibited IP3R3. Activation of IP3R is initiated by IP3 binding to the IBC (IP3-binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP3 binding to the isolated NT (N-terminal; residues 1–604) of IP3R1 and IP3R2, but not to that of IP3R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP3) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP3R activation. IP3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP3R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP3 binding to the chimaeric NT and IP3-evoked Ca2+ release from the chimaeric IP3R. This is the first systematic analysis of the effects of a thiol reagent on each IP3R subtype. We conclude that thimerosal selectively sensitizes IP3R1 and IP3R2 to IP3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor. PMID:23282150

The CW domain, a structural module shared amongst vertebrates, vertebrate-infecting parasites and higher plants.

PubMed

Perry, Jason; Zhao, Yunde

2003-11-01

A previously undetected domain, named CW for its conserved cysteine and tryptophan residues, appears to be a four-cysteine zinc-finger motif found exclusively in vertebrates, vertebrate-infecting parasites and higher plants. Of the twelve distinct nuclear protein families that comprise the CW domain-containing superfamily, only the microrchida (MORC) family has begun to be characterized. However, several families contain other domains suggesting a relationship between the CW domain and either chromatin methylation status or early embryonic development.

Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan-dextran sulfate nanoparticles enhances their expression and anti-cancer effects.

PubMed

Perumal, Vanathi; Arfuso, Frank; Chen, Yan; Fox, Simon; Dharmarajan, Arun M

2018-06-01

In malignant mesothelioma (MM) cells, secreted frizzled-related protein 4 (SFRP4) expression is downregulated by promoter methylation. In this study, we evaluated the effect of encapsulated chitosan-dextran (CS-DS) nanoparticle formulations of SFRP4 and its cysteine-rich domain (CRD) and netrin-like domain (NLD) as means of SFRP4-GFP protein delivery and their effects in JU77 and ONE58 MM cell lines. CS-DS formulations of SFRP4, CRD, and NLD nanoparticles were prepared by a complex coacervation technique, and particle size ranged from 300 nm for empty particles to 337 nm for particles containing the proteins. Measurement of the zeta potential showed that all preparations were around 25 mV or above, suggesting stable formulation and good affinity for the DNA molecules. The CS-DS nanoparticle formulation maintained high integrity and entrapment efficiency. Gene delivery of SFRP4 and its domains showed enhanced biological effects in both JU77 and ONE58 cell lines when compared to the non-liposomal FUGENE ® HD transfection reagent. In comparison to the CRD nanoparticles, both the SFRP4 and NLD nanoparticles significantly reduced the viability of MM cells, with the NLD showing the greatest effect. The CS-DS nanoparticle effects were observed at an earlier time point and with lower DNA concentrations. Morphological changes in MM cells were characterized by the formation of membrane-associated vesicles and green fluorescent protein expression specific to SFRP4 and the NLD. The findings from our proof-of-concept study provide a stepping stone for further investigations using in vivo models.

Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

NASA Technical Reports Server (NTRS)

Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

2016-01-01

Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

PubMed

Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

PubMed Central

Kristensen, Tatjana P.; Maria Cherian, Reeja; Gray, Fiona C.; MacNeill, Stuart A.

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies. PMID:24723920

Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian

2000-01-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

PubMed

Poppers, J; Mulvey, M; Khoo, D; Mohr, I

2000-12-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Analysis of common bean expressed sequence tags identifies sulfur metabolic pathways active in seed and sulfur-rich proteins highly expressed in the absence of phaseolin and major lectins

PubMed Central

2011-01-01

Background A deficiency in phaseolin and phytohemagglutinin is associated with a near doubling of sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris), particularly cysteine, elevated by 70%, and methionine, elevated by 10%. This mostly takes place at the expense of an abundant non-protein amino acid, S-methyl-cysteine. The deficiency in phaseolin and phytohemagglutinin is mainly compensated by increased levels of the 11S globulin legumin and residual lectins. Legumin, albumin-2, defensin and albumin-1 were previously identified as contributing to the increased sulfur amino acid content in the mutant line, on the basis of similarity to proteins from other legumes. Results Profiling of free amino acid in developing seeds of the BAT93 reference genotype revealed a biphasic accumulation of gamma-glutamyl-S-methyl-cysteine, the main soluble form of S-methyl-cysteine, with a lag phase occurring during storage protein accumulation. A collection of 30,147 expressed sequence tags (ESTs) was generated from four developmental stages, corresponding to distinct phases of gamma-glutamyl-S-methyl-cysteine accumulation, and covering the transitions to reserve accumulation and dessication. Analysis of gene ontology categories indicated the occurrence of multiple sulfur metabolic pathways, including all enzymatic activities responsible for sulfate assimilation, de novo cysteine and methionine biosynthesis. Integration of genomic and proteomic data enabled the identification and isolation of cDNAs coding for legumin, albumin-2, defensin D1 and albumin-1A and -B induced in the absence of phaseolin and phytohemagglutinin. Their deduced amino acid sequences have a higher content of cysteine than methionine, providing an explanation for the preferential increase of cysteine in the mutant line. Conclusion The EST collection provides a foundation to further investigate sulfur metabolism and the differential accumulation of sulfur amino acids in seed

KCNQ1 channel modulation by KCNE proteins via the voltage-sensing domain.

PubMed

Nakajo, Koichi; Kubo, Yoshihiro

2015-06-15

The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1-S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR.

PubMed

Chen, Jin-Zhong; Wang, Shu; Tang, Rong; Yang, Quan-Sheng; Zhao, Enpeng; Chao, Yaoqiong; Ying, Kang; Xie, Yi; Mao, Yu-Min

2002-09-01

A cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The 2390 bp cDNA with an open reading fragment (ORF) of 816 bp encodes a 272 amino acids putative protein with a thrombospondin type I repeat (TSR) domain and a cysteine-rich region at the N-terminus, so it is named hPWTSR. We used Northern blot detected two bands with length of about 3 kb and 4 kb respectively, which expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were expressed ubiquitously in fetal tissues and human tumor tissues too. However, the transcript was detected neither in ovarian carcinoma GI-102 nor in lung carcinoma LX-1. Blast analysis against NCBI database revealed that the new gene contained at least 5 exons and located in human chromosome 6q22.33. Our results demonstrate that the gene is a novel member of TSR supergene family.

Identification of ADAM 31: a protein expressed in Leydig cells and specialized epithelia.

PubMed

Liu, L; Smith, J W

2000-06-01

A family of proteins containing a disintegrin and metalloproteinase domain (ADAMs) has been identified recently. Here, we report the identification of a novel member of the ADAM protein family from mouse. This protein is designated ADAM 31. The complementary DNA sequence of ADAM 31 predicts a transmembrane protein with metalloproteinase, disintegrin, cysteine-rich, and cytoplasmic domains. Messenger RNA encoding ADAM 31 was most abundant in testes, but was also detected in many other tissues. More significantly, the antibodies raised against ADAM 31 reveal that the protein has a unique and restricted expression pattern. ADAM 31 is expressed in Leydig cells of the testes, but unlike many other ADAMs, it is not found on developing sperm. Furthermore, ADAM 31 is highly expressed on four types of specialized epithelia: the cauda epididymidis, the vas deferens, the convoluted tubules of the kidney, and the parietal cells of the stomach.

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences

PubMed Central

Nagano, Yukio; Furuhashi, Hirofumi; Inaba, Takehito; Sasaki, Yukiko

2001-01-01

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x2-H-x11-C-x2-C-x(4–5)-C-x2-C-x(3–7)-H-x2-H and C-x2-C-x(10–11)-C-x3-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with 65ZnCl2 showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression. PMID:11600698

Characterization of PhPRP1, a histidine domain arabinogalactan protein from Petunia hybrida pistils.

PubMed

Twomey, Megan C; Brooks, Jenna K; Corey, Jillaine M; Singh-Cundy, Anu

2013-10-15

An arabinogalactan protein, PhPRP1, was purified from Petunia hybrida pistils and shown to be orthologous to TTS-1 and TTS-2 from Nicotiana tabacum and NaTTS from Nicotiana alata. Sequence comparisons among these proteins, and CaPRP1 from Capsicum annuum, reveal a conserved histidine-rich domain and two hypervariable domains. Immunoblots show that TTS-1 and PhPRP1 are also expressed in vegetative tissues of tobacco and petunia respectively. In contrast to the molecular mass heterogeneity displayed by the pistil proteins, the different isoforms found in seedlings, roots, and leaves each has a discrete size (37, 80, 160, and 200 kDa) on SDS-PAGE gels. On the basis of their chemistry, distinctive domain architecture, and the unique pattern of expression, we have named this group of proteins HD-AGPs (histidine domain-arabinogalactan proteins). Copyright © 2013 Elsevier GmbH. All rights reserved.

Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins1[OPEN

PubMed Central

Muzzopappa, Fernando; Wilson, Adjélé; Yogarajah, Vinosa; Cot, Sandrine; Perreau, François; Montigny, Cédric; Bourcier de Carbon, Céline

2017-01-01

The photoactive Orange Carotenoid Protein (OCP) photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain is the active part of the protein, and the C-terminal domain regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins [HCPs]), paralogs of the N-terminal domain of OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized, and they proved to be very good singlet oxygen quenchers. When synthesized in Escherichia coli or Synechocystis PCC 6803, CTDHs formed dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo-CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable to do so. HCPs need the presence of CTDH to become holo-proteins. We propose that, in cyanobacteria, the CTDHs are carotenoid donors to HCPs. PMID:28935842

Protein domain organisation: adding order.

PubMed

Kummerfeld, Sarah K; Teichmann, Sarah A

2009-01-29

Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected degree of clustering and more domain pairs in forward and

Macavirus latency-associated protein evades immune detection through regulation of protein synthesis in cis depending upon its glycin/glutamate-rich domain

PubMed Central

Chen, Ting; Myster, Françoise; Javaux, Justine; Vanderplasschen, Alain

2017-01-01

Alcelaphine herpesvirus 1 (AlHV-1) is a γ-herpesvirus (γ-HV) belonging to the macavirus genus that persistently infects its natural host, the wildebeest, without inducing any clinical sign. However, cross-transmission to other ruminant species causes a deadly lymphoproliferative disease named malignant catarrhal fever (MCF). AlHV-1 ORF73 encodes the latency-associated nuclear antigen (LANA)-homolog protein (aLANA). Recently, aLANA has been shown to be essential for viral persistence in vivo and induction of MCF, suggesting that aLANA shares key properties of other γ-HV genome maintenance proteins. Here we have investigated the evasion of the immune response by aLANA. We found that a glycin/glutamate (GE)-rich repeat domain was sufficient to inhibit in cis the presentation of an epitope linked to aLANA. Although antigen presentation in absence of GE was dependent upon proteasomal degradation of aLANA, a lack of GE did not affect protein turnover. However, protein self-synthesis de novo was downregulated by aLANA GE, a mechanism directly associated with reduced antigen presentation in vitro. Importantly, codon-modification of aLANA GE resulted in increased antigen presentation in vitro and enhanced induction of antigen-specific CD8+ T cell responses in vivo, indicating that mRNA constraints in GE rather than peptidic sequence are responsible for cis-limitation of antigen presentation. Nonetheless, GE-mediated limitation of antigen presentation in cis of aLANA was dispensable during MCF as rabbits developed the disease after virus infection irrespective of the expression of full-length or GE-deficient aLANA. Altogether, we provide evidence that inhibition in cis of protein synthesis through GE is likely involved in long-term immune evasion of AlHV-1 latent persistence in the wildebeest natural host, but dispensable in MCF pathogenesis. PMID:29059246

The length of glycine-rich linker in DNA-binding domain is critical for optimal functioning of quorum-sensing master regulatory protein HapR.

PubMed

Singh, Naorem Santa; Kachhap, Sangita; Singh, Richa; Mishra, Rahul Chandra; Singh, Balvinder; Raychaudhuri, Saumya

2014-12-01

HapR is a quorum-sensing master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of this wild-type protein. While unraveling the underlying cause of functional inertness of a natural variant (HapRV2), the significance of a conserved glycine residue at position 39 in a glycine-rich linker in DNA-binding domain comes into light. This work aims at investigating how the length of glycine-rich linker (R(33)GIGRGG(39)) bridging helices α1 and α2 modulates the functionality of HapR. In pursuit of our interest, glycine residues were inserted after terminal glycine (G39) of the linker in a sequential manner. To evaluate functionality, all the glycine linker variants were subjected to a battery of performance tests under various conditions. Combined in vitro and in vivo results clearly demonstrated a gradual functional impairment of HapR linker variants coupled with increasing length of glycine-rich linker and finally, linker variant harboring four glycine residues resulted in a functionally compromised protein with significant loss of communication with cognate DNAs. Molecular dynamics studies of modeled HapR linker variants in complex with cognate promoter region show that residues namely Ser50, Thr53 and Asn56 are involved in varying degree of interactions with different nucleotides of HapR-DNA complex. The diminished functionality between variants and DNA appears to result from reduced or no interactions between Phe55 and nucleotides of cognate DNA as observed during simulations.

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

PubMed Central

Bocian-Ostrzycka, Katarzyna M.; Łasica, Anna M.; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J.; Drabik, Karolina; Dobosz, Aneta M.; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K.

2015-01-01

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity. PMID:26500620

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

PubMed

Bocian-Ostrzycka, Katarzyna M; Łasica, Anna M; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J; Drabik, Karolina; Dobosz, Aneta M; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K

2015-01-01

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

Hybrid proline-rich proteins: novel players in plant cell elongation?

PubMed Central

Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas

2012-01-01

Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

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

Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this studymore » we show that isolated PH and START domains interact with each other. The crystal structure of a PH–START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH–START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine–rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.« less

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains.

PubMed

Zhou, Huan-Xiang

2006-11-01

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

PubMed

Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

2010-04-01

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

Structural Landscape of the Proline-Rich Domain of Sos1 Nucleotide Exchange Factor

PubMed Central

McDonald, Caleb B.; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C.; Deegan, Brian J.; Seldeen, Kenneth L.; Lednev, Igor K.; Farooq, Amjad

2013-01-01

Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. PMID:23528987

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

PubMed Central

Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

1993-01-01

A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

Murein Lytic Enzyme TgaA of Bifidobacterium bifidum MIMBb75 Modulates Dendritic Cell Maturation through Its Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase (CHAP) Amidase Domain

PubMed Central

Zanoni, Ivan; Balzaretti, Silvia; Miriani, Matteo; Taverniti, Valentina; De Noni, Ivano; Presti, Ilaria; Stuknyte, Milda; Scarafoni, Alessio; Arioli, Stefania; Iametti, Stefania; Bonomi, Francesco; Mora, Diego; Karp, Matti; Granucci, Francesca

2014-01-01

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system. PMID:24814791

Implication of a Central Cysteine Residue and the HHCC Domain of Moloney Murine Leukemia Virus Integrase Protein in Functional Multimerization

PubMed Central

Donzella, George A.; Leon, Oscar; Roth, Monica J.

1998-01-01

Moloney murine leukemia virus (M-MuLV) IN-IN protein interactions important for catalysis of strand transfer and unimolecular and bimolecular disintegration reactions were investigated by using a panel of chemically modified M-MuLV IN proteins. Functional complementation of an HHCC-deleted protein (NΔ105) by an independent HHCC domain (CΔ232) was severely compromised by NEM modification of either subunit. Productive NΔ105 IN-DNA interactions with a disintegration substrate lacking a long terminal repeat 5′-single-stranded tail also required complementation by a functional HHCC domain. Virus encoding the C209A M-MuLV IN mutation exhibited delayed virion production and replication kinetics. PMID:9445080

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

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

Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun

Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein,more » we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.« less

Inference of domain-disease associations from domain-protein, protein-disease and disease-disease relationships.

PubMed

Zhang, Wangshu; Coba, Marcelo P; Sun, Fengzhu

2016-01-11

Protein domains can be viewed as portable units of biological function that defines the functional properties of proteins. Therefore, if a protein is associated with a disease, protein domains might also be associated and define disease endophenotypes. However, knowledge about such domain-disease relationships is rarely available. Thus, identification of domains associated with human diseases would greatly improve our understanding of the mechanism of human complex diseases and further improve the prevention, diagnosis and treatment of these diseases. Based on phenotypic similarities among diseases, we first group diseases into overlapping modules. We then develop a framework to infer associations between domains and diseases through known relationships between diseases and modules, domains and proteins, as well as proteins and disease modules. Different methods including Association, Maximum likelihood estimation (MLE), Domain-disease pair exclusion analysis (DPEA), Bayesian, and Parsimonious explanation (PE) approaches are developed to predict domain-disease associations. We demonstrate the effectiveness of all the five approaches via a series of validation experiments, and show the robustness of the MLE, Bayesian and PE approaches to the involved parameters. We also study the effects of disease modularization in inferring novel domain-disease associations. Through validation, the AUC (Area Under the operating characteristic Curve) scores for Bayesian, MLE, DPEA, PE, and Association approaches are 0.86, 0.84, 0.83, 0.83 and 0.79, respectively, indicating the usefulness of these approaches for predicting domain-disease relationships. Finally, we choose the Bayesian approach to infer domains associated with two common diseases, Crohn's disease and type 2 diabetes. The Bayesian approach has the best performance for the inference of domain-disease relationships. The predicted landscape between domains and diseases provides a more detailed view about the disease

LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.

USDA-ARS?s Scientific Manuscript database

LysK is a staphylococcal bacteriophage endolysin composed of three domains, an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain (cleaves between D-alanine of the stem peptide and glycine of the cross-bridge peptide) a mid-protein amidase 2 domain (N-ace...

The Arabidopsis Cysteine-Rich Receptor-Like Kinase CRK36 Regulates Immunity through Interaction with the Cytoplasmic Kinase BIK1

PubMed Central

Lee, Dong Sook; Kim, Young Cheon; Kwon, Sun Jae; Ryu, Choong-Min; Park, Ohkmae K.

2017-01-01

Receptor-like kinases are important signaling components that regulate a variety of cellular processes. In this study, an Arabidopsis cDNA microarray analysis led to the identification of the cysteine-rich receptor-like kinase CRK36 responsive to the necrotrophic fungal pathogen, Alternaria brassicicola. To determine the function of CRK36 in plant immunity, T-DNA-insertion knockdown (crk36) and overexpressing (CRK36OE) plants were prepared. CRK36OE plants exhibited increased hypersensitive cell death and ROS burst in response to avirulent pathogens. Treatment with a typical pathogen-associated molecular pattern, flg22, markedly induced pattern-triggered immune responses, notably stomatal defense, in CRK36OE plants. The immune responses were weakened in crk36 plants. Protein-protein interaction assays revealed the in vivo association of CRK36, FLS2, and BIK1. CRK36 enhanced flg22-triggered BIK1 phosphorylation, which showed defects with Cys mutations in the DUF26 motifs of CRK36. Disruption of BIK1 and RbohD/RbohF genes further impaired CRK36-mediated stomatal defense. We propose that CRK36, together with BIK1 and NADPH oxidases, may form a positive activation loop that enhances ROS burst and leads to the promotion of stomatal immunity. PMID:29163585

Ribosomal L1 domain and lysine-rich region are essential for CSIG/ RSL1D1 to regulate proliferation and senescence

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

Ma, Liwei; Zhao, Wenting; Zheng, Quanhui

2016-01-15

The expression change of cellular senescence-associated genes is underlying the genetic foundation of cellular senescence. Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) as a novel senescence-associated gene. CSIG is implicated in various process including cell cycle regulation, apoptosis, and tumor metastasis. We previously showed that CSIG plays an important role in regulating cell proliferation and cellular senescence progression through inhibiting PTEN, however, which domain or region of CSIG contributes to this function? To clarify this question, we investigated the functional importance of ribosomal L1 domain and lysine (Lys) -rich region of CSIG. Themore » data showed that expression of CSIG potently reduced PTEN expression, increased cell proliferation rates, and reduced the senescent phenotype (lower SA-β-gal activity). By contrast, neither the expression of CSIG N- terminal (NT) fragment containing the ribosomal L1 domain nor C-terminal (CT) fragment containing Lys-rich region could significantly altered the levels of PTEN; instead of promoting cell proliferation and delaying cellular senescence, expression of CSIG-NT or CSIG-CT inhibited cell proliferation and accelerated cell senescence (increased SA-β-gal activity) compared to either CSIG over-expressing or control (empty vector transfected) cells. The further immunofluorescence analysis showed that CSIG-CT and CSIG-NT truncated proteins exhibited different subcellular distribution with that of wild-type CSIG. Conclusively, both ribosomal L1 domain and Lys-rich region of CSIG are critical for CSIG to act as a regulator of cell proliferation and cellular senescence. - Highlights: • The ribosomal L1 domain and lysine-rich region of CSIG were expressed. • They are critical for CSIG to regulate proliferation and senescence. • CSIG and its domains exhibit different subcellular distribution.« less

Emergence of novel domains in proteins

PubMed Central

2013-01-01

Background Proteins are composed of a combination of discrete, well-defined, sequence domains, associated with specific functions that have arisen at different times during evolutionary history. The emergence of novel domains is related to protein functional diversification and adaptation. But currently little is known about how novel domains arise and how they subsequently evolve. Results To gain insights into the impact of recently emerged domains in protein evolution we have identified all human young protein domains that have emerged in approximately the past 550 million years. We have classified them into vertebrate-specific and mammalian-specific groups, and compared them to older domains. We have found 426 different annotated young domains, totalling 995 domain occurrences, which represent about 12.3% of all human domains. We have observed that 61.3% of them arose in newly formed genes, while the remaining 38.7% are found combined with older domains, and have very likely emerged in the context of a previously existing protein. Young domains are preferentially located at the N-terminus of the protein, indicating that, at least in vertebrates, novel functional sequences often emerge there. Furthermore, young domains show significantly higher non-synonymous to synonymous substitution rates than older domains using human and mouse orthologous sequence comparisons. This is also true when we compare young and old domains located in the same protein, suggesting that recently arisen domains tend to evolve in a less constrained manner than older domains. Conclusions We conclude that proteins tend to gain domains over time, becoming progressively longer. We show that many proteins are made of domains of different age, and that the fastest evolving parts correspond to the domains that have been acquired more recently. PMID:23425224

Click-PEGylation - A mobility shift approach to assess the redox state of cysteines in candidate proteins.

PubMed

van Leeuwen, Lucie A G; Hinchy, Elizabeth C; Murphy, Michael P; Robb, Ellen L; Cochemé, Helena M

2017-07-01

The redox state of cysteine thiols is critical for protein function. Whereas cysteines play an important role in the maintenance of protein structure through the formation of internal disulfides, their nucleophilic thiol groups can become oxidatively modified in response to diverse redox challenges and thereby function in signalling and antioxidant defences. These oxidative modifications occur in response to a range of agents and stimuli, and can lead to the existence of multiple redox states for a given protein. To assess the role(s) of a protein in redox signalling and antioxidant defence, it is thus vital to be able to assess which of the multiple thiol redox states are present and to investigate how these alter under different conditions. While this can be done by a range of mass spectrometric-based methods, these are time-consuming, costly, and best suited to study abundant proteins or to perform an unbiased proteomic screen. One approach that can facilitate a targeted assessment of candidate proteins, as well as proteins that are low in abundance or proteomically challenging, is by electrophoretic mobility shift assays. Redox-modified cysteine residues are selectively tagged with a large group, such as a polyethylene glycol (PEG) polymer, and then the proteins are separated by electrophoresis followed by immunoblotting, which allows the inference of redox changes based on band shifts. However, the applicability of this method has been impaired by the difficulty of cleanly modifying protein thiols by large PEG reagents. To establish a more robust method for redox-selective PEGylation, we have utilised a Click chemistry approach, where free thiol groups are first labelled with a reagent modified to contain an alkyne moiety, which is subsequently Click-reacted with a PEG molecule containing a complementary azide function. This strategy can be adapted to study reversibly reduced or oxidised cysteines. Separation of the thiol labelling step from the PEG

Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines

PubMed Central

Bischak, Connor G.; Longhi, Sonia; Snead, David M.; Costanzo, Stéphanie; Terrer, Elodie; Londergan, Casey H.

2010-01-01

Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) were cyanylated at cysteine and their infrared spectra in the C≡N stretching region were recorded both in the absence and in the presence of one of the physiological partners of NTAIL, namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within NTAIL, the C≡N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level. PMID:20816082

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

PubMed

Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

2012-01-01

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

Modeling Protein Domain Function

ERIC Educational Resources Information Center

Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

2007-01-01

This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

Prioritisation of associations between protein domains and complex diseases using domain-domain interaction networks.

PubMed

Wang, W; Zhang, W; Jiang, R; Luan, Y

2010-05-01

It is of vital importance to find genetic variants that underlie human complex diseases and locate genes that are responsible for these diseases. Since proteins are typically composed of several structural domains, it is reasonable to assume that harmful genetic variants may alter structures of protein domains, affect functions of proteins and eventually cause disorders. With this understanding, the authors explore the possibility of recovering associations between protein domains and complex diseases. The authors define associations between protein domains and disease families on the basis of associations between non-synonymous single nucleotide polymorphisms (nsSNPs) and complex diseases, similarities between diseases, and relations between proteins and domains. Based on a domain-domain interaction network, the authors propose a 'guilt-by-proximity' principle to rank candidate domains according to their average distance to a set of seed domains in the domain-domain interaction network. The authors validate the method through large-scale cross-validation experiments on simulated linkage intervals, random controls and the whole genome. Results show that areas under receiver operating characteristic curves (AUC scores) can be as high as 77.90%, and the mean rank ratios can be as low as 21.82%. The authors further offer a freely accessible web interface for a genome-wide landscape of associations between domains and disease families.

Domain atrophy creates rare cases of functional partial protein domains.

PubMed

Prakash, Ananth; Bateman, Alex

2015-04-30

Protein domains display a range of structural diversity, with numerous additions and deletions of secondary structural elements between related domains. We have observed a small number of cases of surprising large-scale deletions of core elements of structural domains. We propose a new concept called domain atrophy, where protein domains lose a significant number of core structural elements. Here, we implement a new pipeline to systematically identify new cases of domain atrophy across all known protein sequences. The output of this pipeline was carefully checked by hand, which filtered out partial domain instances that were unlikely to represent true domain atrophy due to misannotations or un-annotated sequence fragments. We identify 75 cases of domain atrophy, of which eight cases are found in a three-dimensional protein structure and 67 cases have been inferred based on mapping to a known homologous structure. Domains with structural variations include ancient folds such as the TIM-barrel and Rossmann folds. Most of these domains are observed to show structural loss that does not affect their functional sites. Our analysis has significantly increased the known cases of domain atrophy. We discuss specific instances of domain atrophy and see that there has often been a compensatory mechanism that helps to maintain the stability of the partial domain. Our study indicates that although domain atrophy is an extremely rare phenomenon, protein domains under certain circumstances can tolerate extreme mutations giving rise to partial, but functional, domains.

Methyl-beta-cyclodextrin increases permeability of Caco-2 cell monolayers by displacing specific claudins from cholesterol rich domains associated with tight junctions.

PubMed

Lambert, Daniel; O'Neill, Catherine A; Padfield, Philip J

2007-01-01

In a previous study we demonstrated that depletion of Caco-2 cell cholesterol results in the loss of tight junction (TJ) integrity through the movement of claudins 3 and 4 and occludin, but not claudin 1, out of the TJs [1]. The aims of this study were to determine whether the major tight junction (TJ) proteins in Caco-2 cells are associated with cholesterol rich, membrane raft-like domains and if the loss of TJ integrity produced by the extraction of cholesterol reflects the dissolution of these domains resulting in the loss of TJ organisation. We have demonstrated that in Caco-2 cells claudins 1, 3, 4 and 7, JAM-A and occludin, are associated with cholesterol rich membrane domains that are insoluble in Lubrol WX. Co-immunoprecipitation studies demonstrated that there is no apparent restriction on the combination of claudins present in the rafts and that interaction between the proteins is dependent on cholesterol. JAM-A was not co-immunoprecipitated with the other TJ proteins indicating that it is resident within in a distinct population of rafts and therefore is likely not directly associated with the claudins/occludin present in the TJ complexes. Depletion of Caco-2 cell cholesterol with methyl-beta-cyclodextrin resulted in the displacement of claudins 3, 4 and 7, JAM-A and occludin, but not claudin 1, out of the cholesterol rich domains. Our data indicate that depletion of cholesterol does not result in the loss of the TJ-associated membrane rafts. However, the sterol is required to maintain the association of key proteins with the TJ associated membrane rafts and therefore the TJs. Furthermore, the data suggest that cholesterol may actually directly stabilise the multi-protein complexes that form the TJ strands. Copyright (c) 2007 S. Karger AG, Basel.

Seed storage protein deficiency improves sulfur amino acid content in common bean (Phaseolus vulgaris L.): redirection of sulfur from gamma-glutamyl-S-methyl-cysteine.

PubMed

Taylor, Meghan; Chapman, Ralph; Beyaert, Ronald; Hernández-Sebastià, Cinta; Marsolais, Frédéric

2008-07-23

The contents of sulfur amino acids in seeds of common bean ( Phaseolus vulgaris L.) are suboptimal for nutrition. They accumulate large amounts of a gamma-glutamyl dipeptide of S-methyl-cysteine, a nonprotein amino acid that cannot substitute for methionine or cysteine in the diet. Protein accumulation and amino acid composition were characterized in three genetically related lines integrating a progressive deficiency in major seed storage proteins, phaseolin, phytohemagglutinin, and arcelin. Nitrogen, carbon, and sulfur contents were comparable among the three lines. The contents of S-methyl-cysteine and gamma-glutamyl-S-methyl-cysteine were progressively reduced in the mutants. Sulfur was shifted predominantly to the protein cysteine pool, while total methionine was only slightly elevated. Methionine and cystine contents (mg per g protein) were increased by up to ca. 40%, to levels slightly above FAO guidelines on amino acid requirements for human nutrition. These findings may be useful to improve the nutritional quality of common bean.

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

PubMed Central

Lopez, María V.; Blanco, Patricia; Viale, Diego L.; Cafferata, Eduardo G.; Carbone, Cecilia; Gould, David; Chernajovsky, Yuti; Podhajcer, Osvaldo L.

2009-01-01

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a “cross-talk” between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus-thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells themselves or by coadministered endothelial cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass. PMID:17041094

The Classification of Protein Domains.

PubMed

Dawson, Natalie; Sillitoe, Ian; Marsden, Russell L; Orengo, Christine A

2017-01-01

The significant expansion in protein sequence and structure data that we are now witnessing brings with it a pressing need to bring order to the protein world. Such order enables us to gain insights into the evolution of proteins, their function and the extent to which the functional repertoire can vary across the three kingdoms of life. This has lead to the creation of a wide range of protein family classifications that aim to group proteins based upon their evolutionary relationships.In this chapter we discuss the approaches and methods that are frequently used in the classification of proteins, with a specific emphasis on the classification of protein domains. The construction of both domain sequence and domain structure databases is considered and we show how the use of domain family annotations to assign structural and functional information is enhancing our understanding of genomes.

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

PubMed

Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

Structural landscape of the proline-rich domain of Sos1 nucleotide exchange factor.

PubMed

McDonald, Caleb B; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

2013-01-01

Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. Copyright © 2013 Elsevier B.V. All rights reserved.

On the Dynamical Behavior of the Cysteine Dioxygenase-l-Cysteine Complex in the Presence of Free Dioxygen and l-Cysteine.

PubMed

Pietra, Francesco

2017-11-01

In this work, viable models of cysteine dioxygenase (CDO) and its complex with l-cysteine dianion were built for the first time, under strict adherence to the crystal structure from X-ray diffraction studies, for all atom molecular dynamics (MD). Based on the CHARMM36 FF, the active site, featuring an octahedral dummy Fe(II) model, allowed us observing water exchange, which would have escaped attention with the more popular bonded models. Free dioxygen (O 2 ) and l-cysteine, added at the active site, could be observed being expelled toward the solvating medium under Random Accelerated Molecular Dynamics (RAMD) along major and minor pathways. Correspondingly, free dioxygen (O 2 ), added to the solvating medium, could be observed to follow the same above pathways in getting to the active site under unbiased MD. For the bulky l-cysteine, 600 ns of trajectory were insufficient for protein penetration, and the molecule was stuck at the protein borders. These models pave the way to free energy studies of ligand associations, devised to better clarify how this cardinal enzyme behaves in human metabolism. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Conotoxin Φ-MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti-Apoptotic Activity.

PubMed

Jin, Ai-Hua; Dekan, Zoltan; Smout, Michael J; Wilson, David; Dutertre, Sébastien; Vetter, Irina; Lewis, Richard J; Loukas, Alex; Daly, Norelle L; Alewood, Paul F

2017-11-20

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC 50 17.85 μm) while inhibiting apoptosis (EC 50 2.2 μm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall

PubMed Central

De Caroli, Monica; Lenucci, Marcello S.; Manualdi, Francesca; Dalessandro, Giuseppe; De Lorenzo, Giulia; Piro, Gabriella

2015-01-01

The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D), and C sub-fragments (C1–10), to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required), the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal. PMID:26379688

Multiple graph regularized protein domain ranking.

PubMed

Wang, Jim Jing-Yan; Bensmail, Halima; Gao, Xin

2012-11-19

Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods. To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods. The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications.

The inhibition of calcium carbonate crystal growth by the cysteine-rich Mdm2 peptide.

PubMed

Dalas, E; Chalias, A; Gatos, D; Barlos, K

2006-08-15

The crystal growth of calcite, the most stable calcium carbonate polymorph, in the presence of the cysteine-rich Mdm2 peptide (containing 48 amino acids in the ring finger configuration), has been investigated by the constant composition technique. Crystallization took place exclusively on well-characterized calcite crystals in solutions supersaturated only with respect to this calcium carbonate salt. The kinetic results indicated a surface diffusion spiral growth mechanism. The presence of the Mdm2 peptide inhibited the crystal growth of calcite by 22-58% in the concentration range tested, through adsorption onto the active growth sites of the calcite crystal surface. The kinetic results favored a Langmuir-type adsorption model, and the value of the calculated affinity constant was k(aff)=147x10(4) dm(3)mol(-1), a(ads)=0.29.

Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

PubMed

Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

2010-01-01

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

In vivo inhibition of cysteine proteases provides evidence for the involvement of 'senescence-associated vacuoles' in chloroplast protein degradation during dark-induced senescence of tobacco leaves.

PubMed

Carrión, Cristian A; Costa, María Lorenza; Martínez, Dana E; Mohr, Christina; Humbeck, Klaus; Guiamet, Juan J

2013-11-01

Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction.

PubMed

Naudí, Alba; Jové, Mariona; Cacabelos, Daniel; Ayala, Victoria; Cabre, Rosanna; Caro, Pilar; Gomez, José; Portero-Otín, Manuel; Barja, Gustavo; Pamplona, Reinald

2013-02-01

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N (ε)-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.

The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain

PubMed Central

Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J; Sivakumaran, Andrew; Yoon, Je-Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, B Göran; Wilce, Jacqueline A; Díaz-Moreno, Irene

2014-01-01

T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein–nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5′ TOPs (5′ terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations. PMID:24824036

Changes in rubisco, cysteine-rich proteins and antioxidant system of spinach (Spinacia oleracea L.) due to sulphur deficiency, cadmium stress and their combination.

PubMed

Bagheri, Rita; Ahmad, Javed; Bashir, Humayra; Iqbal, Muhammad; Qureshi, M Irfan

2017-03-01

Sulphur (S) deficiency, cadmium (Cd) toxicity and their combinations are of wide occurrence throughout agricultural lands. We assessed the impact of short-term (2 days) and long-term (4 days) applications of cadmium (40 μg/g soil) on spinach plants grown on sulphur-sufficient (300 μM SO 4 2- ) and sulphur-deficient (30 μM SO 4 2- ) soils. Compared with the control (+S and -Cd), oxidative stress was increased by S deficiency (-S and -Cd), cadmium (+S and +Cd) and their combination stress (-S and +Cd) in the order of (S deficiency) < (Cd stress) < (S deficiency and +Cd stress). SDS-PAGE profile of leaf proteins showed a high vulnerability of rubisco large subunit (RbcL) to S deficiency. Rubisco small subunit (RbcS) was particularly sensitive to Cd as well as dual stress (+Cd and -S) but increased with Cd in the presence of S. Cysteine content in low molecular weight proteins/peptide was also affected, showing a significant increase under cadmium treatment. Components of ascorbate-glutathione antioxidant system altered their levels, showing the maximum decline in ascorbate (ASA), dehydroascorbate (DHA), total ascorbate (ASA + DHA, hereafter TA), glutathione (GSH) and total glutathione (GSH + GSSG, hereafter TG) under S deficiency. However, total ascorbate and total glutathione increased, besides a marginal increase in their reduced and oxidized forms, when Cd was applied in the presence of sufficient S. Sulphur supply also helped in increasing the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) under Cd stress. However, their activity suffered by S deficiency and by Cd stress during S deficiency. Each stress declined the contents of soluble protein and photosynthetic pigments; the highest decline in contents of protein and pigments occurred under S deficiency and dual stress respectively. The fresh and dry weights, although affected adversely by every stress, declined most under dual stress. It

The role of the proline-rich domain of Ssdp1 in the modular architecture of the vertebrate head organizer

PubMed Central

Enkhmandakh, Badam; Makeyev, Alexandr V.; Bayarsaihan, Dashzeveg

2006-01-01

Lim1, Ssdp1, and Ldb1 proteins are components of the Ldb1-associated transcriptional complex, which is important in the head-organizing activity during early mouse development. Depletion of each individual protein alone causes a headless phenotype. To explore in more detail the modular architecture of the complex, we have generated two different gene-trapped mouse lines that express truncated forms of Ssdp1. Embryos derived from the gene-trapped line that encodes a truncated Ssdp1 lacking the proline-rich sequence exhibit a lethal abnormal head-development phenotype, resembling mouse embryos deficient for Lim1, Ssdp1, or Otx2 genes. Embryos derived from the second gene-trapped line, in which most of the proline-rich domain of Ssdp1 is retained, did not show abnormalities in head development. Our data demonstrate that components of the Ldb1-dependent module can be subdivided further into discrete functional domains and that the proline-rich stretch of Ssdp1 is critical for embryonic head development. Furthermore, phylogenetic comparisons revealed that in Caenorhabditis elegans, a similar proline-rich sequence is absent in Ssdp but present in Ldb1. We conclude that although the overall architecture of the Ldb1-dependent module has been preserved, the genetic specification of its individual components has diversified during evolution, without compromising the function of the module. PMID:16864769

The role of the proline-rich domain of Ssdp1 in the modular architecture of the vertebrate head organizer.

PubMed

Enkhmandakh, Badam; Makeyev, Alexandr V; Bayarsaihan, Dashzeveg

2006-08-01

Lim1, Ssdp1, and Ldb1 proteins are components of the Ldb1-associated transcriptional complex, which is important in the head-organizing activity during early mouse development. Depletion of each individual protein alone causes a headless phenotype. To explore in more detail the modular architecture of the complex, we have generated two different gene-trapped mouse lines that express truncated forms of Ssdp1. Embryos derived from the gene-trapped line that encodes a truncated Ssdp1 lacking the proline-rich sequence exhibit a lethal abnormal head-development phenotype, resembling mouse embryos deficient for Lim1, Ssdp1, or Otx2 genes. Embryos derived from the second gene-trapped line, in which most of the proline-rich domain of Ssdp1 is retained, did not show abnormalities in head development. Our data demonstrate that components of the Ldb1-dependent module can be subdivided further into discrete functional domains and that the proline-rich stretch of Ssdp1 is critical for embryonic head development. Furthermore, phylogenetic comparisons revealed that in Caenorhabditis elegans, a similar proline-rich sequence is absent in Ssdp but present in Ldb1. We conclude that although the overall architecture of the Ldb1-dependent module has been preserved, the genetic specification of its individual components has diversified during evolution, without compromising the function of the module.

Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

NASA Astrophysics Data System (ADS)

Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

2013-03-01

Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses

Multiple graph regularized protein domain ranking

PubMed Central

2012-01-01

Background Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods. Results To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods. Conclusion The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications. PMID:23157331

LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

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

Sosa, Brian A.; Rothballer, Andrea; Kutay, Ulrike

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansivemore » grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.« less

Src binds cortactin through an SH2 domain cystine-mediated linkage.

PubMed

Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

2012-12-15

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

Src binds cortactin through an SH2 domain cystine-mediated linkage

PubMed Central

Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

2012-01-01

Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

Proteins in Load-Bearing Junctions: The Histidine-Rich Metal-Binding Protein of Mussel Byssus†,‡

PubMed Central

Zhao, Hua; Waite, J. Herbert

2007-01-01

Building complex load-bearing scaffolds depends on effective ways of joining functionally different biomacromolecules. The junction between collagen fibers and foamlike adhesive plaques in mussel byssus is robust despite the strikingly dissimilar connected structures. mcfp-4, the matrix protein from this junction, and its presecreted form from the foot tissue of Mytilus californianus were isolated and characterized. mcfp-4 has a mass of ∼93 kDa as determined by MALDI-TOF mass spectrometry. Its composition is dominated by histidine (22 mol %), but levels of lysine, arginine, and aspartate are also significant. A small amount of 3,4-dihydroxyphenyl-L-alanine (2 mol %) can be detected by amino acid analysis and redox cycling assays. The cDNA-deduced sequence of mcfp-4 reveals multiple variants with highly repetitive internal structures, including ∼36 tandemly repeated His-rich decapeptides (e.g., HVHTHRVLHK) in the N-terminal half and 16 somewhat more degenerate aspartate-rich undecapeptides (e.g., DDHVNDIAQTA) in the C-terminal half. Incubation of a synthetic peptide based on the His-rich decapeptide with Fe3+, Co2+, Ni2+, Zn2+, and Cu2+ indicates that only Cu is strongly bound. MALDI-TOF mass spectrometry of the peptide modified with diethyl pyrocarbonate before and after Cu binding suggests that histidine residues dominate Cu binding. In contrast, the aspartate-rich undecapeptides preferentially bind Ca2+. mcfp-4 is strategically positioned to function as a macromolecular bifunctional linker by using metal ions to couple its own His-rich domains to the His-rich termini of the preCOLs. Ca2+ may mediate coupling of the C-terminus to other calcium-binding plaque proteins. PMID:17115717

Genetic selection for a highly functional cysteine-less membrane protein using site-saturation mutagenesis

PubMed Central

Arendt, Cassandra S.; Ri, Keirei; Yates, Phillip A.; Ullman, Buddy

2007-01-01

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site-saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2, in order to facilitate biochemical studies using thiol-specific modifying reagents. Of ten endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site-saturation mutagenesis scheme based on the Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site-saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position. PMID:17481563

The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein

PubMed Central

Qin, Wenxia; Wunderley, Lydia; Barrett, Anne L.; High, Stephen; Woodman, Philip G.

2016-01-01

LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins. PMID:27582497

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

PubMed

Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

2014-11-01

Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

The Dictyostelium Carmil Protein Links Capping Protein and the Arp2/3 Complex to Type I Myosins through Their Sh3 Domains

PubMed Central

Jung, Goeh; Remmert, Kirsten; Wu, Xufeng; Volosky, Joanne M.; III, John A. Hammer

2001-01-01

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the α and β subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end–directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans

A thermodynamic definition of protein domains.

PubMed

Porter, Lauren L; Rose, George D

2012-06-12

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.

Targeting cysteine-mediated dimerization of the MUC1-C oncoprotein in human cancer cells

PubMed Central

RAINA, DEEPAK; AHMAD, REHAN; RAJABI, HASAN; PANCHAMOORTHY, GOVIND; KHARBANDA, SURENDER; KUFE, DONALD

2012-01-01

The MUC1 heterodimeric protein is aberrantly overexpressed in diverse human carcinomas and contributes to the malignant phenotype. The MUC1-C transmembrane subunit contains a CQC motif in the cytoplasmic domain that has been implicated in the formation of dimers and in its oncogenic function. The present study demonstrates that MUC1-C forms dimers in human breast and lung cancer cells. MUC1-C dimerization was detectable in the cytoplasm and was independent of MUC1-N, the N-terminal mucin subunit that extends outside the cell. We show that the MUC1-C cytoplasmic domain forms dimers in vitro that are disrupted by reducing agents. Moreover, dimerization of the MUC1-C subunit in cancer cells was blocked by reducing agents and increased by oxidative stress, supporting involvement of the CQC motif in forming disulfide bonds. In support of these observations, mutation of the MUC1-C CQC motif to AQA completely blocked MUC1-C dimerization. Importantly, this study was performed with MUC1-C devoid of fluorescent proteins, such as GFP, CFP and YFP. In this regard, we show that GFP, CFP and YFP themselves form dimers that are readily detectable with cross-linking agents. The present results further demonstrate that a cell-penetrating peptide that targets the MUC1-C CQC cysteines blocks MUC1-C dimerization in cancer cells. These findings provide definitive evidence that: i) the MUC1-C cytoplasmic domain cysteines are necessary and sufficient for MUC1-C dimerization, and ii) these CQC motif cysteines represent an Achilles’ heel for targeting MUC1-C function. PMID:22200620

Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis; Implications for Vaccine Development

PubMed Central

van de Waterbeemd, Bas; Zomer, Gijsbert; van den IJssel, Jan; van Keulen, Lonneke; Eppink, Michel H.; van der Ley, Peter; van der Pol, Leo A.

2013-01-01

Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis. PMID:23372704

Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

PubMed

Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

1992-05-15

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and

Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

PubMed Central

Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

1992-01-01

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and

Mapping and Engineering Functional Domains of the Assembly Activating Protein of Adeno-Associated Viruses.

PubMed

Tse, Longping V; Moller-Tank, Sven; Meganck, Rita M; Asokan, Aravind

2018-04-25

Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis followed by deletion and substitutional mutagenesis of specific domains, namely, the hydrophobic N-terminal domain (HR), conserved core (CC), proline-rich region (PRR), threonine/serine rich region (T/S) and basic region (BR). First, we establish that the centrally located PRR and T/S regions are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display varying ability to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N-terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful towards understanding and controlling AAV capsid assembly. Importance: Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Understanding how AAP acts as a chaperone for viral assembly could help improve efficiency and potentially control this process. Our studies reveal that AAP has a modular architecture, with each module playing a

Cellular Localization and Characterization of Cytosolic Binding Partners for Gla Domain-containing Proteins PRRG4 and PRRG2*

PubMed Central

Yazicioglu, Mustafa N.; Monaldini, Luca; Chu, Kirk; Khazi, Fayaz R.; Murphy, Samuel L.; Huang, Heshu; Margaritis, Paris; High, Katherine A.

2013-01-01

The genes encoding a family of proteins termed proline-rich γ-carboxyglutamic acid (PRRG) proteins were identified and characterized more than a decade ago, but their functions remain unknown. These novel membrane proteins have an extracellular γ-carboxyglutamic acid (Gla) protein domain and cytosolic WW binding motifs. We screened WW domain arrays for cytosolic binding partners for PRRG4 and identified novel protein-protein interactions for the protein. We also uncovered a new WW binding motif in PRRG4 that is essential for these newly found protein-protein interactions. Several of the PRRG-interacting proteins we identified are essential for a variety of physiologic processes. Our findings indicate possible novel and previously unidentified functions for PRRG proteins. PMID:23873930

J domain independent functions of J proteins.

PubMed

Ajit Tamadaddi, Chetana; Sahi, Chandan

2016-07-01

Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not "completely" rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes.

Ginkgotides: Proline-Rich Hevein-Like Peptides from Gymnosperm Ginkgo biloba.

PubMed

Wong, Ka H; Tan, Wei Liang; Serra, Aida; Xiao, Tianshu; Sze, Siu Kwan; Yang, Daiwen; Tam, James P

2016-01-01

Hevein and hevein-like peptides belong to the family of chitin-binding cysteine-rich peptides. They are classified into three subfamilies, the prototypic 8C- and the 6C- and 10C-hevein-like peptides. Thus far, only five 8C-hevein-like peptides have been characterized from three angiosperms and none from gymnosperm. To determine their occurrence and distribution in the gymnosperm, Ginkgo biloba leaves were examined. Here, we report the discovery and characterization of 11 novel 8C-hevein-like peptides, namely ginkgotides gB1-gB11. Proteomic analysis showed that the ginkgotides contain 41-44 amino acids (aa), a chitin-binding domain and are Pro-rich, a distinguishing feature that differs from other hevein-like peptides. Solution NMR structure determination revealed that gB5 contains a three β-stranded structure shaped by a cystine knot with an additional disulfide bond at the C-terminus. Transcriptomic analysis showed that the ginkgotide precursors contain a three-domain architecture, comprised of a C-terminal tail (20 aa) that is significantly shorter than those of other 8C- and 10C-hevein-like peptides, which generally contain a protein cargo such as a Barwin-like protein (126 aa) or class I chitinase (254 aa). Transcriptomic data mining found an additional 48 ginkgotide homologs in 39 different gymnosperms. Phylogenetic analysis revealed that ginkgotides and their homologs belong to a new class of 8C-hevein-like peptides. Stability studies showed that ginkgotides are highly resistant to thermal, acidic and endopeptidase degradation. Ginkgotides flanked at both the N- and C-terminal ends by Pro were resistant to exopeptidase degradation by carboxypeptidase A and aminopeptidase. Antifungal assays showed that ginkgotides inhibit the hyphal growth of phyto-pathogenic fungi. Taken together, ginkgotides represent the first suite of hevein-like peptides isolated and characterized from gymnosperms. As a group, they represent a novel class of 8C-hevein-like peptides that

A Genetic Screen Identifies a Requirement for Cysteine-Rich-Receptor-Like Kinases in Rice NH1 (OsNPR1)-Mediated Immunity.

PubMed

Chern, Mawsheng; Xu, Qiufang; Bart, Rebecca S; Bai, Wei; Ruan, Deling; Sze-To, Wing Hoi; Canlas, Patrick E; Jain, Rashmi; Chen, Xuewei; Ronald, Pamela C

2016-05-01

Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases (CRK6 and CRK10), complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1 phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. These experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression.

The ANGULATA7 gene encodes a DnaJ-like zinc finger-domain protein involved in chloroplast function and leaf development in Arabidopsis.

PubMed

Muñoz-Nortes, Tamara; Pérez-Pérez, José Manuel; Ponce, María Rosa; Candela, Héctor; Micol, José Luis

2017-03-01

The characterization of mutants with altered leaf shape and pigmentation has previously allowed the identification of nuclear genes that encode plastid-localized proteins that perform essential functions in leaf growth and development. A large-scale screen previously allowed us to isolate ethyl methanesulfonate-induced mutants with small rosettes and pale green leaves with prominent marginal teeth, which were assigned to a phenotypic class that we dubbed Angulata. The molecular characterization of the 12 genes assigned to this phenotypic class should help us to advance our understanding of the still poorly understood relationship between chloroplast biogenesis and leaf morphogenesis. In this article, we report the phenotypic and molecular characterization of the angulata7-1 (anu7-1) mutant of Arabidopsis thaliana, which we found to be a hypomorphic allele of the EMB2737 gene, which was previously known only for its embryonic-lethal mutations. ANU7 encodes a plant-specific protein that contains a domain similar to the central cysteine-rich domain of DnaJ proteins. The observed genetic interaction of anu7-1 with a loss-of-function allele of GENOMES UNCOUPLED1 suggests that the anu7-1 mutation triggers a retrograde signal that leads to changes in the expression of many genes that normally function in the chloroplasts. Many such genes are expressed at higher levels in anu7-1 rosettes, with a significant overrepresentation of those required for the expression of plastid genome genes. Like in other mutants with altered expression of plastid-encoded genes, we found that anu7-1 exhibits defects in the arrangement of thylakoidal membranes, which appear locally unappressed. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

The synthesis and protein resistance of amphiphilic PDMS-b-(PDMS-g-cysteine) copolymers

NASA Astrophysics Data System (ADS)

Lei, Yufeng; Lin, Yaling; Zhang, Anqiang

2017-10-01

Zwitterionic polymers have been used to cope with nonspecific protein adsorption and bio-fouling problems for a wide range of materials, including biomedical devices, marine coatings and membrane separation. However, direct surface modification with highly water-soluble zwitterionic polymers is rather difficult due to their poor attachment to hydrophobic solid surfaces. In this work, we utilize the hydrophobic interaction to anchor zwitterionic polysiloxanes grafted with cysteine onto surfaces by adding an hydrophobic block of polydimethylsiloxanes, referred as PDMS-b-(PDMS-g-Cys)s. The synthesis involves only three steps of reactions, and the structures of each product were characterized using GPC, FT-IR and 1H NMR. The adsorption and protein resistance of PDMS-b-(PDMS-g-Cys)s on a gold surface are investigated with QCM-D. The results show that the hydrophobic interaction moieties of the additional PDMS blocks help the hydrophilic cysteine-grafted blocks stably attach and then function on the sensor. These findings suggest that the addition of hydrophobic moieties provides an effective approach to construct anti-fouling interfaces with zwitterionic polymers in aqueous solution.

A novel Toll like receptor with two TIR domains (HcToll-2) is involved in regulation of antimicrobial peptide gene expression of Hyriopsis cumingii.

PubMed

Ren, Qian; Lan, Jiang-Feng; Zhong, Xue; Song, Xiao-Jun; Ma, Fei; Hui, Kai-Min; Wang, Wen; Yu, Xiao-Qiang; Wang, Jin-Xing

2014-07-01

Animal Toll-like receptors (TLRs) are involved in innate immunity. Toll proteins are generally transmembrane proteins. In this study, an atypical Toll-like receptor (HcToll-2) was identified from the triangle-shell pearl mussel Hyriopsis cumingii, which belongs to phylum Mollusca. Unlike the typical Toll like receptors with extracellular leucine-rich repeats (LRRs), transmembrane, and intracellular Toll/interleukin-1 receptor (TIR) domains, HcToll-2 has two homologous TIR domains located at the C-terminal (designated as HcTIR1 and HcTIR2) and lacks a transmembrane domain. Phylogenetic analysis showed that HcTIR1 was clustered with TIR of sea anemone Toll, and HcTIR2 was clustered with TIR of Drosophila Toll. HcToll-2 mRNA could be detected in the hepatopancreas and was upregulated after challenge with Escherichia coli and Staphylococcus aureus. Recombinant HcLRR protein with GST tag could bind to bacteria and also to LPS and PGN. Over-expression of both HcTIR1 and HcTIR2 induced drosomycin genes in Drosophila S2 cells. RNAi analysis showed that HcToll-2 was required for the expression of theromacin, which is a cysteine-rich antimicrobial peptide (AMP) gene. This research is the first report of an atypical Toll-like receptor HcToll-2 involved in antibacterial immunity through induction of AMP expression. Copyright © 2014 Elsevier Ltd. All rights reserved.

Occurrence, Functions and Biological Significance of Arginine-Rich Proteins.

PubMed

Chandana, Thimmegowda; Venkatesh, Yeldur P

2016-01-01

Arginine, the most basic among the 20 amino acids, occurs less frequently than lysine in proteins despite being coded by six codons. Only a few important proteins of biological significance have been found to be abundant in arginine. It has been established that these arginine-rich proteins have been assigned important roles in the biological systems. Arginine-rich cationic proteins are known to stabilize macromolecular structures by establishing appropriate interactions (salt bridges, hydrogen bonds and cation-π interactions). These proteins are also known to be the key members of many regulatory pathways such as gene expression, chromatin stability, expurgation of introns from naïve mRNA, mRNA splicing, membrane-penetrating activity and pathogenesis-related defense, to name a few. Further, arginine occurs in various combinations with other amino acids (serine, lysine, proline, tryptophan, valine, glycine and glutamic acid) which diversify the potential functions of arginine-rich proteins. Arginine-rich proteins known till date from dietary sources have been described in terms of their structure and functional properties. A variety of activities such as bactericidal, membrane-penetrating, antimicrobial, anti-hypertensive, pro-angiogenic and others have been reported for arginine-rich proteins. This review attempts to collate the occurrence, functions and the biological significance of this unique class of proteins rich in arginine.

L-cysteine suppresses ghrelin and reduces appetite in rodents and humans.

PubMed

McGavigan, A K; O'Hara, H C; Amin, A; Kinsey-Jones, J; Spreckley, E; Alamshah, A; Agahi, A; Banks, K; France, R; Hyberg, G; Wong, C; Bewick, G A; Gardiner, J V; Lehmann, A; Martin, N M; Ghatei, M A; Bloom, S R; Murphy, K G

2015-03-01

High-protein diets promote weight loss and subsequent weight maintenance, but are difficult to adhere to. The mechanisms by which protein exerts these effects remain unclear. However, the amino acids produced by protein digestion may have a role in driving protein-induced satiety. We tested the effects of a range of amino acids on food intake in rodents and identified l-cysteine as the most anorexigenic. Using rodents we further studied the effect of l-cysteine on food intake, behaviour and energy expenditure. We proceeded to investigate its effect on neuronal activation in the hypothalamus and brainstem before investigating its effect on gastric emptying and gut hormone release. The effect of l-cysteine on appetite scores and gut hormone release was then investigated in humans. l-Cysteine dose-dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This effect did not appear to be secondary to behavioural or aversive side effects. l-Cysteine increased neuronal activation in the area postrema and delayed gastric emptying. It suppressed plasma acyl ghrelin levels and did not reduce food intake in transgenic ghrelin-overexpressing mice. Repeated l-cysteine administration decreased food intake in rats and obese mice. l-Cysteine reduced hunger and plasma acyl ghrelin levels in humans. Further work is required to determine the chronic effect of l-cysteine in rodents and humans on appetite and body weight, and whether l-cysteine contributes towards protein-induced satiety.

l-cysteine suppresses ghrelin and reduces appetite in rodents and humans

PubMed Central

McGavigan, A K; O'Hara, H C; Amin, A; Kinsey-Jones, J; Spreckley, E; Alamshah, A; Agahi, A; Banks, K; France, R; Hyberg, G; Wong, C; Bewick, G A; Gardiner, J V; Lehmann, A; Martin, N M; Ghatei, M A; Bloom, S R; Murphy, K G

2015-01-01

Background: High-protein diets promote weight loss and subsequent weight maintenance, but are difficult to adhere to. The mechanisms by which protein exerts these effects remain unclear. However, the amino acids produced by protein digestion may have a role in driving protein-induced satiety. Methods: We tested the effects of a range of amino acids on food intake in rodents and identified l-cysteine as the most anorexigenic. Using rodents we further studied the effect of l-cysteine on food intake, behaviour and energy expenditure. We proceeded to investigate its effect on neuronal activation in the hypothalamus and brainstem before investigating its effect on gastric emptying and gut hormone release. The effect of l-cysteine on appetite scores and gut hormone release was then investigated in humans. Results: l-Cysteine dose-dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This effect did not appear to be secondary to behavioural or aversive side effects. l-Cysteine increased neuronal activation in the area postrema and delayed gastric emptying. It suppressed plasma acyl ghrelin levels and did not reduce food intake in transgenic ghrelin-overexpressing mice. Repeated l-cysteine administration decreased food intake in rats and obese mice. l-Cysteine reduced hunger and plasma acyl ghrelin levels in humans. Conclusions: Further work is required to determine the chronic effect of l-cysteine in rodents and humans on appetite and body weight, and whether l-cysteine contributes towards protein-induced satiety. PMID:25219528

Structure and DNA-Binding Sites of the SWI1 AT-rich Interaction Domain (ARID) Suggest Determinants for Sequence-Specific DNA Recognition

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

Kim, Suhkmann; Zhang, Ziming; Upchurch, Sean

2004-04-16

2 ARID is a homologous family of DNA-binding domains that occur in DNA binding proteins from a wide variety of species, ranging from yeast to nematodes, insects, mammals and plants. SWI1, a member of the SWI/SNF protein complex that is involved in chromatin remodeling during transcription, contains the ARID motif. The ARID domain of human SWI1 (also known as p270) does not select for a specific DNA sequence from a random sequence pool. The lack of sequence specificity shown by the SWI1 ARID domain stands in contrast to the other characterized ARID domains, which recognize specific AT-rich sequences. We havemore » solved the three-dimensional structure of human SWI1 ARID using solution NMR methods. In addition, we have characterized non-specific DNA-binding by the SWI1 ARID domain. Results from this study indicate that a flexible long internal loop in ARID motif is likely to be important for sequence specific DNA-recognition. The structure of human SWI1 ARID domain also represents a distinct structural subfamily. Studies of ARID indicate that boundary of the DNA binding structural and functional domains can extend beyond the sequence homologous region in a homologous family of proteins. Structural studies of homologous domains such as ARID family of DNA-binding domains should provide information to better predict the boundary of structural and functional domains in structural genomic studies. Key Words: ARID, SWI1, NMR, structural genomics, protein-DNA interaction.« less

Wheat glutenin: the "tail" of the 1By protein subunits.

PubMed

Nunes-Miranda, Júlio D; Bancel, Emmanuelle; Viala, Didier; Chambon, Christophe; Capelo, José L; Branlard, Gérard; Ravel, Catherine; Igrejas, Gilberto

2017-10-03

Gluten-forming storage proteins play a major role in the viscoelastic properties of wheat dough through the formation of a continuous proteinaceous network. The high-molecular-weight glutenin subunits represent a functionally important subgroup of gluten proteins by promoting the formation of large glutenin polymers through interchain disulphide bonds between glutenin subunits. Here, we present evidences that y-type glutenin subunits encoded at the Glu-B1 locus are prone to proteolytic processing at the C-terminus tail, leading to the loss of the unique cysteine residue present at the C-terminal domain. Results obtained by intact mass measurement and immunochemistry for each proteoform indicate that the proteolytic cleavage appears to occur at the carboxyl-side of two conserved asparagine residues at the C-terminal domain start. Hence, we hypothesize that the responsible enzymes are a class of cysteine endopeptidases - asparaginyl endopeptidases - described in post-translational processing of other storage proteins in wheat. Biological significance The reported study provides new insights into wheat storage protein maturation. In view of the importance of gluten proteins on dough viscoelastic properties and end-product quality, the reported C-terminal domain cleavage of high-molecular-weight glutenin subunits is of particular interest, since this domain possesses a unique conserved cysteine residue which is assumed to participate in gluten polymerization. Copyright © 2017 Elsevier B.V. All rights reserved.

The LINE-1 DNA sequences in four mammalian orders predict proteins that conserve homologies to retrovirus proteins.

PubMed Central

Fanning, T; Singer, M

1987-01-01

Recent work suggests that one or more members of the highly repeated LINE-1 (L1) DNA family found in all mammals may encode one or more proteins. Here we report the sequence of a portion of an L1 cloned from the domestic cat (Felis catus). These data permit comparison of the L1 sequences in four mammalian orders (Carnivore, Lagomorph, Rodent and Primate) and the comparison supports the suggested coding potential. In two separate, noncontiguous regions in the carboxy terminal half of the proteins predicted from the DNA sequences, there are several strongly conserved segments. In one region, these share homology with known or suspected reverse transcriptases, as described by others in rodents and primates. In the second region, closer to the carboxy terminus, the strongly conserved segments are over 90% homologous among the four orders. One of the latter segments is cysteine rich and resembles the putative metal binding domains of nucleic acid binding proteins, including those of TFIIIA and retroviruses. PMID:3562227

Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

PubMed

Abraham, Anup; Chandler, Douglas E

2017-10-01

Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

PubMed

Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

2017-02-26

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs. Copyright © 2017 Elsevier Inc. All rights reserved.

Forced Unfolding of Proteins Within Cells

PubMed Central

Johnson, Colin P.; Tang, Hsin-Yao; Carag, Christine; Speicher, David W.; Discher, Dennis E.

2009-01-01

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells—as a prototypical adherent cell—nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to colocalize signaling events such as phosphorylation with forced unfolding. PMID:17673662

Same but not alike: Structure, flexibility and energetics of domains in multi-domain proteins are influenced by the presence of other domains

PubMed Central

Vishwanath, Sneha

2018-01-01

The majority of the proteins encoded in the genomes of eukaryotes contain more than one domain. Reasons for high prevalence of multi-domain proteins in various organisms have been attributed to higher stability and functional and folding advantages over single-domain proteins. Despite these advantages, many proteins are composed of only one domain while their homologous domains are part of multi-domain proteins. In the study presented here, differences in the properties of protein domains in single-domain and multi-domain systems and their influence on functions are discussed. We studied 20 pairs of identical protein domains, which were crystallized in two forms (a) tethered to other proteins domains and (b) tethered to fewer protein domains than (a) or not tethered to any protein domain. Results suggest that tethering of domains in multi-domain proteins influences the structural, dynamic and energetic properties of the constituent protein domains. 50% of the protein domain pairs show significant structural deviations while 90% of the protein domain pairs show differences in dynamics and 12% of the residues show differences in the energetics. To gain further insights on the influence of tethering on the function of the domains, 4 pairs of homologous protein domains, where one of them is a full-length single-domain protein and the other protein domain is a part of a multi-domain protein, were studied. Analyses showed that identical and structurally equivalent functional residues show differential dynamics in homologous protein domains; though comparable dynamics between in-silico generated chimera protein and multi-domain proteins were observed. From these observations, the differences observed in the functions of homologous proteins could be attributed to the presence of tethered domain. Overall, we conclude that tethered domains in multi-domain proteins not only provide stability or folding advantages but also influence pathways resulting in differences in

Same but not alike: Structure, flexibility and energetics of domains in multi-domain proteins are influenced by the presence of other domains.

PubMed

Vishwanath, Sneha; de Brevern, Alexandre G; Srinivasan, Narayanaswamy

2018-02-01

The majority of the proteins encoded in the genomes of eukaryotes contain more than one domain. Reasons for high prevalence of multi-domain proteins in various organisms have been attributed to higher stability and functional and folding advantages over single-domain proteins. Despite these advantages, many proteins are composed of only one domain while their homologous domains are part of multi-domain proteins. In the study presented here, differences in the properties of protein domains in single-domain and multi-domain systems and their influence on functions are discussed. We studied 20 pairs of identical protein domains, which were crystallized in two forms (a) tethered to other proteins domains and (b) tethered to fewer protein domains than (a) or not tethered to any protein domain. Results suggest that tethering of domains in multi-domain proteins influences the structural, dynamic and energetic properties of the constituent protein domains. 50% of the protein domain pairs show significant structural deviations while 90% of the protein domain pairs show differences in dynamics and 12% of the residues show differences in the energetics. To gain further insights on the influence of tethering on the function of the domains, 4 pairs of homologous protein domains, where one of them is a full-length single-domain protein and the other protein domain is a part of a multi-domain protein, were studied. Analyses showed that identical and structurally equivalent functional residues show differential dynamics in homologous protein domains; though comparable dynamics between in-silico generated chimera protein and multi-domain proteins were observed. From these observations, the differences observed in the functions of homologous proteins could be attributed to the presence of tethered domain. Overall, we conclude that tethered domains in multi-domain proteins not only provide stability or folding advantages but also influence pathways resulting in differences in

Proteolysis of noncollagenous proteins in sea cucumber, Stichopus japonicus, body wall: characterisation and the effects of cysteine protease inhibitors.

PubMed

Wu, Hai-Tao; Li, Dong-Mei; Zhu, Bei-Wei; Sun, Jin-Jian; Zheng, Jie; Wang, Feng-Lin; Konno, Kunihiko; Jiang, Xi

2013-11-15

Proteolysis of noncollagenous proteins in sea cucumber, Stichopus Japonicus, body wall (sjBW) was investigated. The proteins removed from sjBW by SDS and urea extraction were mainly noncollagenous proteins with molecular weights about 200kDa (Band I) and 44kDa (Band II), respectively. Band I and Band II were identified as major yolk protein (MYP) and actin, respectively, from holothurian species by liquid chromatography-mass spectrometry (LC-MS/MS) with significant scores. Based on TCA-soluble oligopeptide assay, the optimum proteolysis condition of noncollagenous proteins was at 46.3°C and pH 6.1, by response surface methodology. The proteolysis of MYP, and actin, was partially inhibited by cysteine protease inhibitors, including Trans-epoxysuccinyl-l-leucyl-amido (4-guanidino) butane (E-64), iodoacetic acid, antipain and whey protein concentrate. These results suggest that cysteine proteases are partially involved in the proteolysis of noncollagenous proteins in body wall of sea cucumber, S. japonicus. Copyright © 2013 Elsevier Ltd. All rights reserved.

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration*

PubMed Central

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W.

2014-01-01

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions. PMID:24532791

Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

PubMed

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W

2014-03-28

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

Transcriptome Analysis Revealed Highly Expressed Genes Encoding Secondary Metabolite Pathways and Small Cysteine-Rich Proteins in the Sclerotium of Lignosus rhinocerotis

PubMed Central

Yap, Hui-Yeng Y.; Chooi, Yit-Heng; Fung, Shin-Yee; Ng, Szu-Ting; Tan, Chon-Seng; Tan, Nget-Hong

2015-01-01

Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications. PMID:26606395

Rethinking Cysteine Protective Groups: S-Alkylsulfonyl-l-Cysteines for Chemoselective Disulfide Formation.

PubMed

Schäfer, Olga; Huesmann, David; Muhl, Christian; Barz, Matthias

2016-12-12

The ability to reversibly cross-link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S-alkylsulfonyl-l-cysteines and N-carboxy anhydrides (NCA) thereof for peptide synthesis. The S-alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well-defined homo- as well as block co-polymers. Yet, thiols react immediately with the S-alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Composition, Protein Profile and Rheological Properties of Pseudocereal-Based Protein-Rich Ingredients.

PubMed

Alonso-Miravalles, Loreto; O'Mahony, James A

2018-05-07

The objectives of this study were to investigate the nutrient composition, protein profile, morphology, and pasting properties of protein-rich pseudocereal ingredients (quinoa, amaranth, and buckwheat) and compare them to the more common rice and maize flours. Literature concerning protein-rich pseudocereal ingredients is very limited, mainly to protein profiling. The concentrations of macronutrients (i.e., ash, fat, and protein, as well as soluble, insoluble and total dietary fibre) were significantly higher for the protein-rich variants of pseudocereal-based flours than their regular protein content variants and the rice and maize flours. On profiling the protein component using sodium dodecyl sulfate⁻polyacrylamide gel electrophoresis (SDS-PAGE), all samples showed common bands at ~50 kDa and low molecular weight bands corresponding to the globulin fraction (~50 kDa) and albumin fraction (~10 kDa), respectively; except rice, in which the main protein was glutelin. The morphology of the starch granules was studied using scanning electron microscopy with quinoa and amaranth showing the smallest sized granules, while buckwheat, rice, and maize had the largest starch granules. The pasting properties of the ingredients were generally similar, except for buckwheat and amaranth, which showed the highest and lowest final viscosity, respectively. The results obtained in this study can be used to better understand the functionality and food applications of protein-rich pseudocereal ingredients.

AIDA: ab initio domain assembly for automated multi-domain protein structure prediction and domain–domain interaction prediction

PubMed Central

Xu, Dong; Jaroszewski, Lukasz; Li, Zhanwen; Godzik, Adam

2015-01-01

Motivation: Most proteins consist of multiple domains, independent structural and evolutionary units that are often reshuffled in genomic rearrangements to form new protein architectures. Template-based modeling methods can often detect homologous templates for individual domains, but templates that could be used to model the entire query protein are often not available. Results: We have developed a fast docking algorithm ab initio domain assembly (AIDA) for assembling multi-domain protein structures, guided by the ab initio folding potential. This approach can be extended to discontinuous domains (i.e. domains with ‘inserted’ domains). When tested on experimentally solved structures of multi-domain proteins, the relative domain positions were accurately found among top 5000 models in 86% of cases. AIDA server can use domain assignments provided by the user or predict them from the provided sequence. The latter approach is particularly useful for automated protein structure prediction servers. The blind test consisting of 95 CASP10 targets shows that domain boundaries could be successfully determined for 97% of targets. Availability and implementation: The AIDA package as well as the benchmark sets used here are available for download at http://ffas.burnham.org/AIDA/. Contact: adam@sanfordburnham.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25701568

Predicting protein-protein interactions from protein domains using a set cover approach.

PubMed

Huang, Chengbang; Morcos, Faruck; Kanaan, Simon P; Wuchty, Stefan; Chen, Danny Z; Izaguirre, Jesús A

2007-01-01

One goal of contemporary proteome research is the elucidation of cellular protein interactions. Based on currently available protein-protein interaction and domain data, we introduce a novel method, Maximum Specificity Set Cover (MSSC), for the prediction of protein-protein interactions. In our approach, we map the relationship between interactions of proteins and their corresponding domain architectures to a generalized weighted set cover problem. The application of a greedy algorithm provides sets of domain interactions which explain the presence of protein interactions to the largest degree of specificity. Utilizing domain and protein interaction data of S. cerevisiae, MSSC enables prediction of previously unknown protein interactions, links that are well supported by a high tendency of coexpression and functional homogeneity of the corresponding proteins. Focusing on concrete examples, we show that MSSC reliably predicts protein interactions in well-studied molecular systems, such as the 26S proteasome and RNA polymerase II of S. cerevisiae. We also show that the quality of the predictions is comparable to the Maximum Likelihood Estimation while MSSC is faster. This new algorithm and all data sets used are accessible through a Web portal at http://ppi.cse.nd.edu.

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase.

PubMed

Mehrotra, Sonali; B Ningappa, Mylarappa; Raman, Jayalakshmi; Anand, Ranjith P; Balaram, Hemalatha

2012-04-01

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion. Copyright © 2012 Elsevier B.V. All rights reserved.

Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

PubMed Central

2010-01-01

Background Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII), which is the most variable segment of the protein. Methods To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure. Results The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii) PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. Conclusions This study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion. PMID:21092207

How Egg Case Proteins Can Protect Cuttlefish Offspring?

PubMed Central

Cornet, Valérie; Henry, Joël; Goux, Didier; Duval, Emilie; Bernay, Benoit; Le Corguillé, Gildas; Corre, Erwan; Zatylny-Gaudin, Céline

2015-01-01

Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection. PMID:26168161

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Keratinization-associated miR-7 and miR-21 Regulate Tumor Suppressor Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK) in Oral Cancer*

PubMed Central

Jung, Hyun Min; Phillips, Brittany L.; Patel, Rushi S.; Cohen, Donald M.; Jakymiw, Andrew; Kong, William W.; Cheng, Jin Q.; Chan, Edward K. L.

2012-01-01

MicroRNAs (miRNAs) are small non-coding RNAs that posttranscriptionally regulate gene expression during many biological processes. Recently, the aberrant expressions of miRNAs have become a major focus in cancer research. The purpose of this study was to identify deregulated miRNAs in oral cancer and further focus on specific miRNAs that were related to patient survival. Here, we report that miRNA expression profiling provided more precise information when oral squamous cell carcinomas were subcategorized on the basis of clinicopathological parameters (tumor primary site, histological subtype, tumor stage, and HPV16 status). An innovative radar chart analysis method was developed to depict subcategories of cancers taking into consideration the expression patterns of multiple miRNAs combined with the clinicopathological parameters. Keratinization of tumors and the high expression of miR-21 were the major factors related to the poor prognosis of patients. Interestingly, a majority of the keratinized tumors expressed high levels of miR-21. Further investigations demonstrated the regulation of the tumor suppressor gene reversion-inducing cysteine-rich protein with kazal motifs (RECK) by two keratinization-associated miRNAs, miR-7 and miR-21. Transfection of miR-7 and miR-21-mimics reduced the expression of RECK through direct miRNA-mediated regulation, and these miRNAs were inversely correlated with RECK in CAL 27 orthotopic xenograft tumors. Furthermore, a similar inverse correlation was demonstrated in CAL 27 cells treated in vitro by different external stimuli such as trypsinization, cell density, and serum concentration. Taken together, our data show that keratinization is associated with poor prognosis of oral cancer patients and keratinization-associated miRNAs mediate deregulation of RECK which may contribute to the aggressiveness of tumors. PMID:22761427

Reconstituting protein interaction networks using parameter-dependent domain-domain interactions

PubMed Central

2013-01-01

Background We can describe protein-protein interactions (PPIs) as sets of distinct domain-domain interactions (DDIs) that mediate the physical interactions between proteins. Experimental data confirm that DDIs are more consistent than their corresponding PPIs, lending support to the notion that analyses of DDIs may improve our understanding of PPIs and lead to further insights into cellular function, disease, and evolution. However, currently available experimental DDI data cover only a small fraction of all existing PPIs and, in the absence of structural data, determining which particular DDI mediates any given PPI is a challenge. Results We present two contributions to the field of domain interaction analysis. First, we introduce a novel computational strategy to merge domain annotation data from multiple databases. We show that when we merged yeast domain annotations from six annotation databases we increased the average number of domains per protein from 1.05 to 2.44, bringing it closer to the estimated average value of 3. Second, we introduce a novel computational method, parameter-dependent DDI selection (PADDS), which, given a set of PPIs, extracts a small set of domain pairs that can reconstruct the original set of protein interactions, while attempting to minimize false positives. Based on a set of PPIs from multiple organisms, our method extracted 27% more experimentally detected DDIs than existing computational approaches. Conclusions We have provided a method to merge domain annotation data from multiple sources, ensuring large and consistent domain annotation for any given organism. Moreover, we provided a method to extract a small set of DDIs from the underlying set of PPIs and we showed that, in contrast to existing approaches, our method was not biased towards DDIs with low or high occurrence counts. Finally, we used these two methods to highlight the influence of the underlying annotation density on the characteristics of extracted DDIs. Although

Evolutionary dynamics of protein domain architecture in plants

PubMed Central

2012-01-01

Background Protein domains are the structural, functional and evolutionary units of the protein. Protein domain architectures are the linear arrangements of domain(s) in individual proteins. Although the evolutionary history of protein domain architecture has been extensively studied in microorganisms, the evolutionary dynamics of domain architecture in the plant kingdom remains largely undefined. To address this question, we analyzed the lineage-based protein domain architecture content in 14 completed green plant genomes. Results Our analyses show that all 14 plant genomes maintain similar distributions of species-specific, single-domain, and multi-domain architectures. Approximately 65% of plant domain architectures are universally present in all plant lineages, while the remaining architectures are lineage-specific. Clear examples are seen of both the loss and gain of specific protein architectures in higher plants. There has been a dynamic, lineage-wise expansion of domain architectures during plant evolution. The data suggest that this expansion can be largely explained by changes in nuclear ploidy resulting from rounds of whole genome duplications. Indeed, there has been a decrease in the number of unique domain architectures when the genomes were normalized into a presumed ancestral genome that has not undergone whole genome duplications. Conclusions Our data show the conservation of universal domain architectures in all available plant genomes, indicating the presence of an evolutionarily conserved, core set of protein components. However, the occurrence of lineage-specific domain architectures indicates that domain architecture diversity has been maintained beyond these core components in plant genomes. Although several features of genome-wide domain architecture content are conserved in plants, the data clearly demonstrate lineage-wise, progressive changes and expansions of individual protein domain architectures, reinforcing the notion that plant

Prediction of redox-sensitive cysteines using sequential distance and other sequence-based features.

PubMed

Sun, Ming-An; Zhang, Qing; Wang, Yejun; Ge, Wei; Guo, Dianjing

2016-08-24

Reactive oxygen species can modify the structure and function of proteins and may also act as important signaling molecules in various cellular processes. Cysteine thiol groups of proteins are particularly susceptible to oxidation. Meanwhile, their reversible oxidation is of critical roles for redox regulation and signaling. Recently, several computational tools have been developed for predicting redox-sensitive cysteines; however, those methods either only focus on catalytic redox-sensitive cysteines in thiol oxidoreductases, or heavily depend on protein structural data, thus cannot be widely used. In this study, we analyzed various sequence-based features potentially related to cysteine redox-sensitivity, and identified three types of features for efficient computational prediction of redox-sensitive cysteines. These features are: sequential distance to the nearby cysteines, PSSM profile and predicted secondary structure of flanking residues. After further feature selection using SVM-RFE, we developed Redox-Sensitive Cysteine Predictor (RSCP), a SVM based classifier for redox-sensitive cysteine prediction using primary sequence only. Using 10-fold cross-validation on RSC758 dataset, the accuracy, sensitivity, specificity, MCC and AUC were estimated as 0.679, 0.602, 0.756, 0.362 and 0.727, respectively. When evaluated using 10-fold cross-validation with BALOSCTdb dataset which has structure information, the model achieved performance comparable to current structure-based method. Further validation using an independent dataset indicates it is robust and of relatively better accuracy for predicting redox-sensitive cysteines from non-enzyme proteins. In this study, we developed a sequence-based classifier for predicting redox-sensitive cysteines. The major advantage of this method is that it does not rely on protein structure data, which ensures more extensive application compared to other current implementations. Accurate prediction of redox-sensitive cysteines not

The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2β, Are Crucial to Autoantibody Binding in Type 1 Diabetes

PubMed Central

Elvers, Karen T.; Geoghegan, Ivey; Shoemark, Debbie K.; Lampasona, Vito; Bingley, Polly J.; Williams, Alistair J.K.

2013-01-01

Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2β also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2β resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2β PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions. PMID:22966073

Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica

PubMed Central

Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Watanabe, Haruo

2014-01-01

ABSTRACT l-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled l-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of l-cysteine in E. histolytica. [U-13C3, 15N]l-cysteine was rapidly metabolized into three unknown metabolites, besides l-cystine and l-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of l-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of l-cysteine. Liberation of l-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these l-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress. PMID:25370494

Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Sugar beet (Beta vulgaris L.) PGIP genes, BvPGIP1, BvPGIP2 and BvPGIP3, were isolated from two breeding lines, F1016 and F1010. Full-length cDNA sequences of the three BvPGIP genes encod...

Storage proteins of common bean identified with 2D-PAGE

USDA-ARS?s Scientific Manuscript database

The common bean is a significant source of protein, complex carbohydrates, fiber, and minerals. Seeds of most dry beans contain 15 to 25% protein and are rich in lysine but low in the sulfur containing amino acids cysteine and methionine. Knowledge of common bean proteins is important for research a...

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets

PubMed Central

Guzmán Prieto, Ana M.; Urbanus, Rolf T.; Zhang, Xinglin; Bierschenk, Damien; Koekman, C. Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P.; Pape, Marieke; Paganelli, Fernanda L.; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P. A.; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

2015-01-01

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets. PMID:26675410

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

PubMed

Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

2015-12-17

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

Structure of the OsSERK2 leucine-rich repeat extracellular domain.

PubMed

McAndrew, Ryan; Pruitt, Rory N; Kamita, Shizuo G; Pereira, Jose Henrique; Majumdar, Dipali; Hammock, Bruce D; Adams, Paul D; Ronald, Pamela C

2014-11-01

Somatic embryogenesis receptor kinases (SERKs) are leucine-rich repeat (LRR)-containing integral membrane receptors that are involved in the regulation of development and immune responses in plants. It has recently been shown that rice SERK2 (OsSERK2) is essential for XA21-mediated resistance to the pathogen Xanthomonas oryzae pv. oryzae. OsSERK2 is also required for the BRI1-mediated, FLS2-mediated and EFR-mediated responses to brassinosteroids, flagellin and elongation factor Tu (EF-Tu), respectively. Here, crystal structures of the LRR domains of OsSERK2 and a D128N OsSERK2 mutant, expressed as hagfish variable lymphocyte receptor (VLR) fusions, are reported. These structures suggest that the aspartate mutation does not generate any significant conformational change in the protein, but instead leads to an altered interaction with partner receptors.

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein-coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro.

PubMed

Yeh, Szu-Tsen; Zambrano, Cristina M; Koch, Walter J; Purcell, Nicole H

2018-05-25

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Basis for Regulated Proteolysis by the α-Secretase ADAM10.

PubMed

Seegar, Tom C M; Killingsworth, Lauren B; Saha, Nayanendu; Meyer, Peter A; Patra, Dhabaleswar; Zimmerman, Brandon; Janes, Peter W; Rubinstein, Eric; Nikolov, Dimitar B; Skiniotis, Georgios; Kruse, Andrew C; Blacklow, Stephen C

2017-12-14

Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of a consensus protein domain dictionary

PubMed Central

Schaeffer, R. Dustin; Jonsson, Amanda L.; Simms, Andrew M.; Daggett, Valerie

2011-01-01

Motivation: The discovery of new protein folds is a relatively rare occurrence even as the rate of protein structure determination increases. This rarity reinforces the concept of folds as reusable units of structure and function shared by diverse proteins. If the folding mechanism of proteins is largely determined by their topology, then the folding pathways of members of existing folds could encompass the full set used by globular protein domains. Results: We have used recent versions of three common protein domain dictionaries (SCOP, CATH and Dali) to generate a consensus domain dictionary (CDD). Surprisingly, 40% of the metafolds in the CDD are not composed of autonomous structural domains, i.e. they are not plausible independent folding units. This finding has serious ramifications for bioinformatics studies mining these domain dictionaries for globular protein properties. However, our main purpose in deriving this CDD was to generate an updated CDD to choose targets for MD simulation as part of our dynameomics effort, which aims to simulate the native and unfolding pathways of representatives of all globular protein consensus folds (metafolds). Consequently, we also compiled a list of representative protein targets of each metafold in the CDD. Availability and implementation: This domain dictionary is available at www.dynameomics.org. Contact: daggett@u.washington.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21068000

π-Clamp-mediated cysteine conjugation

NASA Astrophysics Data System (ADS)

Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

2016-02-01

Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

Co-evolutionary Analysis of Domains in Interacting Proteins Reveals Insights into Domain–Domain Interactions Mediating Protein–Protein Interactions

PubMed Central

Jothi, Raja; Cherukuri, Praveen F.; Tasneem, Asba; Przytycka, Teresa M.

2006-01-01

Recent advances in functional genomics have helped generate large-scale high-throughput protein interaction data. Such networks, though extremely valuable towards molecular level understanding of cells, do not provide any direct information about the regions (domains) in the proteins that mediate the interaction. Here, we performed co-evolutionary analysis of domains in interacting proteins in order to understand the degree of co-evolution of interacting and non-interacting domains. Using a combination of sequence and structural analysis, we analyzed protein–protein interactions in F1-ATPase, Sec23p/Sec24p, DNA-directed RNA polymerase and nuclear pore complexes, and found that interacting domain pair(s) for a given interaction exhibits higher level of co-evolution than the noninteracting domain pairs. Motivated by this finding, we developed a computational method to test the generality of the observed trend, and to predict large-scale domain–domain interactions. Given a protein–protein interaction, the proposed method predicts the domain pair(s) that is most likely to mediate the protein interaction. We applied this method on the yeast interactome to predict domain–domain interactions, and used known domain–domain interactions found in PDB crystal structures to validate our predictions. Our results show that the prediction accuracy of the proposed method is statistically significant. Comparison of our prediction results with those from two other methods reveals that only a fraction of predictions are shared by all the three methods, indicating that the proposed method can detect known interactions missed by other methods. We believe that the proposed method can be used with other methods to help identify previously unrecognized domain–domain interactions on a genome scale, and could potentially help reduce the search space for identifying interaction sites. PMID:16949097

Expression, refolding and bio-structural analysis of a tetravalent recombinant dengue envelope domain III protein for serological diagnosis.

PubMed

Combe, Maxime; Lacoux, Xavier; Martinez, Jérôme; Méjan, Odile; Luciani, Françoise; Daniel, Soizic

2017-05-01

Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of β-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections. Copyright © 2017 Elsevier Inc. All rights reserved.

A site-directed mutagenesis analysis of tNOX functional domains

NASA Technical Reports Server (NTRS)

Chueh, Pin-Ju; Morre, Dorothy M.; Morre, D. James

2002-01-01

Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

PubMed

Merabet, Samir; Litim-Mecheri, Isma; Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

2011-10-01

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

PubMed Central

Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K.; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

2011-01-01

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. PMID:22046139

Gluten gel and film properties in the presence of cysteine and sodium alginate.

PubMed

Yuno-Ohta, Naoko; Yamada, Mariko; Inomata, Masako; Konagai, Hiromi; Kataoka, Tomomi

2009-08-01

Wheat flour has an ability of forming dough by mixing with water, which exhibits a rheological property required for making bread. The major protein is gluten, which is a valuable protein material for food industry. In this study, gluten protein gels and films were formed with cysteine and sodium alginate. Adding cysteine improved gel and film properties (stress relaxation behavior, bending strength). The gel containing 0.01 M cysteine had a longer relaxation time and was more rigid than the gel without cysteine. Although adding sodium alginate to the gluten suspension containing cysteine improved the water-holding ability and homogeneity of the gel network, the film from this gel was more brittle than the gluten film with cysteine alone. Microstructural observations of the gels and films with scanning electron microscopy suggested that water evaporation was more heterogeneous from the gel containing sodium alginate than from the gel with cysteine alone. Fourier transform-infrared (FT-IR) analysis during film formation suggested that the presence of cysteine encourages interaction between gluten molecules and results in intermolecular beta-sheet formation in earlier stages than in the no additive condition. FT-IR results also suggested that the combined effect of sodium alginate and cysteine on the protein secondary structure was remarkably different from that of cysteine alone. Our results suggest that addition of a suitable amount of cysteine (0.01 M) and heat treatment to 80 degrees C during gluten gel and film formation induces a homogenous network in the gel and film by regulating disulfide-sulfide interactions.

Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins.

PubMed

Chaikam, Vijay; Karlson, Dale T

2010-01-01

The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs. [BMB reports 2010; 43(1): 1-8].

Ginkgotides: Proline-Rich Hevein-Like Peptides from Gymnosperm Ginkgo biloba

PubMed Central

Wong, Ka H.; Tan, Wei Liang; Serra, Aida; Xiao, Tianshu; Sze, Siu Kwan; Yang, Daiwen; Tam, James P.

2016-01-01

Hevein and hevein-like peptides belong to the family of chitin-binding cysteine-rich peptides. They are classified into three subfamilies, the prototypic 8C- and the 6C- and 10C-hevein-like peptides. Thus far, only five 8C-hevein-like peptides have been characterized from three angiosperms and none from gymnosperm. To determine their occurrence and distribution in the gymnosperm, Ginkgo biloba leaves were examined. Here, we report the discovery and characterization of 11 novel 8C-hevein-like peptides, namely ginkgotides gB1–gB11. Proteomic analysis showed that the ginkgotides contain 41–44 amino acids (aa), a chitin-binding domain and are Pro-rich, a distinguishing feature that differs from other hevein-like peptides. Solution NMR structure determination revealed that gB5 contains a three β-stranded structure shaped by a cystine knot with an additional disulfide bond at the C-terminus. Transcriptomic analysis showed that the ginkgotide precursors contain a three-domain architecture, comprised of a C-terminal tail (20 aa) that is significantly shorter than those of other 8C- and 10C-hevein-like peptides, which generally contain a protein cargo such as a Barwin-like protein (126 aa) or class I chitinase (254 aa). Transcriptomic data mining found an additional 48 ginkgotide homologs in 39 different gymnosperms. Phylogenetic analysis revealed that ginkgotides and their homologs belong to a new class of 8C-hevein-like peptides. Stability studies showed that ginkgotides are highly resistant to thermal, acidic and endopeptidase degradation. Ginkgotides flanked at both the N- and C-terminal ends by Pro were resistant to exopeptidase degradation by carboxypeptidase A and aminopeptidase. Antifungal assays showed that ginkgotides inhibit the hyphal growth of phyto-pathogenic fungi. Taken together, ginkgotides represent the first suite of hevein-like peptides isolated and characterized from gymnosperms. As a group, they represent a novel class of 8C-hevein-like peptides

Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

PubMed

Righino, Benedetta; Pirolli, Davide; Radicioni, Giorgia; Marzano, Valeria; Longhi, Renato; Arcovito, Alessandro; Sanna, Maria Teresa; De Rosa, Maria Cristina; Paoluzi, Serena; Cesareni, Gianni; Messana, Irene; Castagnola, Massimo; Vitali, Alberto

2016-09-01

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016. © 2016 Wiley Periodicals, Inc.

Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

PubMed

Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

2006-12-01

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

Intriguing olfactory proteins from the yellow fever mosquito, Aedes aegypti

NASA Astrophysics Data System (ADS)

Ishida, Yuko; Chen, Angela M.; Tsuruda, Jennifer M.; Cornel, Anthon J.; Debboun, Mustapha; Leal, Walter S.

2004-09-01

Four antennae-specific proteins (AaegOBP1, AaegOBP2, AaegOBP3, and AaegASP1) were isolated from the yellow fever mosquito, Aedes aegypti and their full-length cDNAs were cloned. RT-PCR indicated that they are expressed in female and, to a lesser extent, in male antennae, but not in control tissues (legs). AaegOBP1 and AaegOBP3 showed significant similarity to previously identified mosquito odorant-binding proteins (OBPs) in cysteine spacing pattern and sequence. Two of the isolated proteins have a total of eight cysteine residues. The similarity of the spacing pattern of the cysteine residues and amino acid sequence to those of previously identified olfactory proteins suggests that one of the cysteine-rich proteins (AaegOBP2) is an OBP. The other (AaegASP1) did not belong to any group of known OBPs. Structural analyses indicate that six of the cysteine residues in AaegOBP2 are linked in a similar pattern to the previously known cysteine pairing in OBPs, i.e., Cys-24 Cys-55, Cys-51 Cys-104, Cys-95 Cys-113. The additional disulfide bridge, Cys-38 Cys-125, knits the extended C-terminal segment of the protein to a predicted α2-helix. As indicated by circular dichroism (CD) spectra, the extra rigidity seems to prevent the predicted formation of a C-terminal α-helix at low pH.

Resolution of Site-Specific Conformational Heterogeneity in Proline-Rich Molecular Recognition by Src Homology 3 Domains.

PubMed

Horness, Rachel E; Basom, Edward J; Mayer, John P; Thielges, Megan C

2016-02-03

Conformational heterogeneity and dynamics are increasingly evoked in models of protein molecular recognition but are challenging to experimentally characterize. Here we combine the inherent temporal resolution of infrared (IR) spectroscopy with the spatial resolution afforded by selective incorporation of carbon-deuterium (C-D) bonds, which provide frequency-resolved absorptions within a protein IR spectrum, to characterize the molecular recognition of the Src homology 3 (SH3) domain of the yeast protein Sho1 with its cognate proline-rich (PR) sequence of Pbs2. The IR absorptions of C-D bonds introduced at residues along a peptide of the Pbs2 PR sequence report on the changes in the local environments upon binding to the SH3 domain. Interestingly, upon forming the complex the IR spectra of the peptides labeled with C-D bonds at either of the two conserved prolines of the PXXP consensus recognition sequence show more absorptions than there are C-D bonds, providing evidence for the population of multiple states. In contrast, the NMR spectra of the peptides labeled with (13)C at the same residues show only single resonances, indicating rapid interconversion on the NMR time scale. Thus, the data suggest that the SH3 domain recognizes its cognate peptide with a component of induced fit molecular recognition involving the adoption of multiples states, which have previously gone undetected due to interconversion between the populated states that is too fast to resolve using conventional methods.