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1.
Subtilisin E (SbtE) is a member of the ubiquitous superfamily of serine proteases called subtilases and serves as a model for understanding propeptide-mediated protein folding mechanisms. Unlike most proteins that adopt thermodynamically stable conformations, the native state of SbtE is trapped into a kinetically stable conformation. While kinetic stability offers distinct functional advantages to the native state, the constraints that dictate the selection between kinetic and thermodynamic folding and stability remain unknown. Using highly conserved subtilases, we demonstrate that adaptive evolution of sequence dictates selection of folding pathways. Intracellular and extracellular serine proteases (ISPs and ESPs, respectively) constitute two subfamilies within the family of subtilases that have highly conserved sequences, structures, and catalytic activities. Our studies on the folding pathways of subtilisin E (SbtE), an ESP, and its homologue intracellular serine protease 1 (ISP1), an ISP, show that although topology, contact order, and hydrophobicity that drive protein folding reactions are conserved, ISP1 and SbtE fold through significantly different pathways and kinetics. While SbtE absolutely requires the propeptide to fold into a kinetically trapped conformer, ISP1 folds to a thermodynamically stable state more than 1 million times faster and independent of a propeptide. Furthermore, kinetics establish that ISP1 and SbtE fold through different intermediate states. An evolutionary analysis of folding constraints in subtilases suggests that observed differences in folding pathways may be mediated through positive selection of specific residues that map mostly onto the protein surface. Together, our results demonstrate that closely related subtilases can fold through distinct pathways and mechanisms, and suggest that fine sequence details can dictate the choice between kinetic and thermodynamic folding and stability. 相似文献
2.
Adeline Heurich Heike Hofmann-Winkler Stefanie Gierer Thomas Liepold Olaf Jahn Stefan P?hlmann 《Journal of virology》2014,88(2):1293-1307
The type II transmembrane serine proteases TMPRSS2 and HAT can cleave and activate the spike protein (S) of the severe acute respiratory syndrome coronavirus (SARS-CoV) for membrane fusion. In addition, these proteases cleave the viral receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), and it was proposed that ACE2 cleavage augments viral infectivity. However, no mechanistic insights into this process were obtained and the relevance of ACE2 cleavage for SARS-CoV S protein (SARS-S) activation has not been determined. Here, we show that arginine and lysine residues within ACE2 amino acids 697 to 716 are essential for cleavage by TMPRSS2 and HAT and that ACE2 processing is required for augmentation of SARS-S-driven entry by these proteases. In contrast, ACE2 cleavage was dispensable for activation of the viral S protein. Expression of TMPRSS2 increased cellular uptake of soluble SARS-S, suggesting that protease-dependent augmentation of viral entry might be due to increased uptake of virions into target cells. Finally, TMPRSS2 was found to compete with the metalloprotease ADAM17 for ACE2 processing, but only cleavage by TMPRSS2 resulted in augmented SARS-S-driven entry. Collectively, our results in conjunction with those of previous studies indicate that TMPRSS2 and potentially related proteases promote SARS-CoV entry by two separate mechanisms: ACE2 cleavage, which might promote viral uptake, and SARS-S cleavage, which activates the S protein for membrane fusion. These observations have interesting implications for the development of novel therapeutics. In addition, they should spur efforts to determine whether receptor cleavage promotes entry of other coronaviruses, which use peptidases as entry receptors. 相似文献
3.
Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms 总被引:7,自引:0,他引:7
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Porter DC Zhang N Danes C McGahren MJ Harwell RM Faruki S Keyomarsi K 《Molecular and cellular biology》2001,21(18):6254-6269
Cyclin E is a G(1) cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G(1)-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G(2)/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein. 相似文献
4.
Christina Krß Petra Engele Bernhard Sprenger Andreas Fischer Nico Lingg Magdalena Baier Christoph
hlknecht Bettina Lier Chris Oostenbrink Monika Cserjan-Puschmann Gerald Striedner Alois Jungbauer Rainer Schneider 《The Journal of biological chemistry》2021,297(4)
Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner. 相似文献
5.
Dongsheng Xu Aini Wan Lin Peng Yun Chen Yang He Jianfeng Yang 《Preparative biochemistry & biotechnology》2017,47(5):489-495
Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12?mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins. 相似文献
6.
BACKGROUND: Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli. RESULTS: We used the beta-galactosidase alpha-complementation system to monitor and evolve two antibody fragments for high expression levels in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>107 clones), clones exhibiting high levels of beta-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the beta-galactosidase activity present in the cells. CONCLUSION: This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free alpha-peptide released from the protein fusion by the host proteases. This means that the alpha-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released alpha-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein. 相似文献
7.
Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight β-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica. 相似文献
8.
Amal Ben Amira Julien Bauwens Edwin De Pauw Souhail Besbes Hamadi Attia Frédéric Francis Christophe Blecker 《Journal of chemical biology》2017,10(1):25-33
Proteomic approach was applied to identify total proteins, particularly the enzymatic content, from wild cardoon flowers. As the selection of an appropriate sample preparation method is the key for getting reliable results, two different extraction/precipitation methods (trichloroacetic acid and phenol/ammonium acetate) were tested on fresh and lyophilized flowers. After two-dimensional electrophoresis (2D–E) separations, a better protein pattern was obtained after phenol extraction from lyophilized flowers. Only 46 % of the total analyzed spots resulted in a protein identification by mass spectrometry MALDI-TOF. Four proteases (cardosins A, E, G, and H), which have become a subject of great interest in dairy technology, were identified. They presented molecular weights and isoelectric points very close and high levels of homology between matched peptides sequences. The absence of the other cardosins (B, C, D, and F) could be an advantage, as it reduces the excessive proteolytic activity that causes bitter flavors and texture defects, during cheese making. 相似文献
9.
HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases
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The role of proteases in pathogenesis is well established for several microorganisms but has not been described for Yersinia enterocolitica. Previously, we identified a gene, hreP, which showed significant similarity to proteases in a screen for chromosomal genes of Y. enterocolitica that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like inv, the gene encoding the invasin protein of Y. enterocolitica, hreP is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genomic organization of this chromosomal region is different in Escherichia coli, Salmonella, and Yersinia pestis than in Y. enterocolitica. Analysis of the distribution of hreP between different Yersinia isolates and the relatively low G+C content of the gene suggests acquisition by horizontal gene transfer. Sequence analysis also revealed that HreP belongs to a family of eukaryotic subtilisin/kexin-like proteases. Together with the calcium-dependent protease PrcA of Anabaena variabilis, HreP forms a new subfamily of bacterial subtilisin/kexin-like proteases which might have originated from a common eukaryotic ancestor. Like other proteases of this family, HreP is expressed with an N-terminal prosequence. Expression of an HreP-His(6) tag fusion protein in E. coli revealed that HreP undergoes autocatalytic processing at a consensus cleavage site of subtilisin/kexin-like proteases, thereby releasing the proprotein. 相似文献
10.
Escherichia coli is a clonal species. The best-understood components of its clonal variation are the flagellar (H) and polysaccharide (O) antigens, both well documented since the mid-1930s because of their use in serotyping. Flagellin is the protein subunit of the flagellum that carries H-antigen specificity. We show that 43 of the 54 H-antigen specificities of E. coli map to the flagellin gene at fliC and sequenced all 43 forms and confirmed specificity of each by cloning and expression. This is, to our knowledge, the first time that all known forms of such a highly polymorphic gene have been fully sequenced and characterized for any species. The established distinction between a highly variable central region and more conserved flanking regions is upheld. The sequences fall into two groups, one of which may be derived from the fliC gene of the E. coli/Salmonella enterica common ancestor, the other perhaps obtained by lateral transfer since species divergence. Comparison of sequences revealed that both horizontal DNA transfer and fixation of mutations under diversifying selection pressure contributed to polymorphism in this locus. 相似文献
11.
Two high-molecular-mass proteases have been detected in E.coli K12 and isolated from the periplasmic fraction released by osmotic shock. The two proteases, designated Protease peri7 and Protease peri8, have similar molecular masses (greater than 2000 kDa) and degrade alpha- and beta-casein, but not insulin B chain. Protease peri7 is a metalloprotease activated 3-6 fold by ATP, dATP and GTP but inhibited by AMP. Nucleotide hydrolysis occurs during protein breakdown. Protease peri8, in contrast, is a serine protease unaffected by nucleotides or metal chelators. The two proteases appear by electron microscopy to be ring-shaped particles of approximately 125 A degrees in diameter. These proteases appear to be very similar to the multi-protease complexes (Proteasomes) detected in a variety of eukaryotic cells. 相似文献
12.
Edward Emmott Trevor R. Sweeney Ian Goodfellow 《The Journal of biological chemistry》2015,290(46):27841-27853
The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6–7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present. 相似文献
13.
14.
Rose CJ Chapman JR Marshall SD Lee SF Batterham P Ross HA Newcomb RD 《Molecular biology and evolution》2011,28(6):1835-1846
A major theoretical consequence of selection at a locus is the genetic hitchhiking of linked sites (selective sweep). The extent of hitchhiking around a gene is related to the strength of selection and the rate of recombination, with its impact diminishing with distance from the selected site. At the Rop-1 locus of the sheep blowfly, Lucilia cuprina, polymorphisms at two different sites within the LcαE7 gene encode forms of the protein that confer organophosphorus insecticide resistance. To assess the impact of selection at these two sites on variation around LcαE7, we sequenced regions within six other genes along chromosome IV across isogenic (IV) strains of L. cuprina. High levels of linkage disequilibrium, characterized by low haplotype number (K) and diversity (H), and significant R(2) values were observed for two genes, LcαE1 and LcαE10, both members of the same α-esterase gene cluster as LcαE7. A significant R(2) value was also observed for a gene predicted to be the next closest to LcαE7, AL03, but not for any of the other genes, LcRpL13a, Lcdsx, or LcAce. Skews in the site frequency spectra toward high-frequency variants were significant for LcαE1 (Fay and Wu's H = -2.91), LcαE10 (H = -1.85), and Lcdsx (H = -2.00). Since the selective sweeps, two forms of likely returning variation were observed, including variation in microsatellites in an intron of LcαE10 and a recombination event between LcαE7 and LcαE10. These data suggest that two incomplete soft sweeps have occurred at LcαE7 that have significantly affected variation across, and beyond, the α-esterase gene cluster of L. cuprina. The speed and impact of these selective sweeps on surrounding genomic variation and the ability of L. cuprina to respond to future environmental challenges are discussed. 相似文献
15.
alpha2-Macroglobulin (alpha2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), and carries beta-amyloid peptide. alpha2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant alpha2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the alpha2M subunit. However, because intact alpha2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the alpha2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length alpha2M. These mutations did not perturb the tetrameric structure of alpha2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact alpha2M. E730R did not alter TGF-beta1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-beta1 binding associated with alpha2M conformational change. These studies demonstrate that growth factor binding to intact alpha2M is specific, involving a defined region of the alpha2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences. 相似文献
16.
Intramembrane proteases hydrolyze peptide bonds within the membrane as a signaling paradigm universal to all life forms and with implications in disease. Deciphering the architectural strategies supporting intramembrane proteolysis is an essential but unattained goal. We integrated new, quantitative and high-throughput thermal light-scattering technology, reversible equilibrium unfolding and refolding and quantitative protease assays to interrogate rhomboid architecture with 151 purified variants. Rhomboid proteases maintain low intrinsic thermodynamic stability (ΔG = 2.1-4.5 kcal mol(-1)) resulting from a multitude of generally weak transmembrane packing interactions, making them highly responsive to their environment. Stability is consolidated by two buried glycines and several packing leucines, with a few multifaceted hydrogen bonds strategically deployed to two peripheral regions. Opposite these regions lie transmembrane segment 5 and connected loops that are notably exempt of structural responsibility, suggesting intramembrane proteolysis involves considerable but localized protein dynamics. Our analyses provide a comprehensive 'heat map' of the physiochemical anatomy underlying membrane-immersed enzyme function at, what is to our knowledge, unprecedented resolution. 相似文献
17.
A protein of the Z class of liver cytosolic proteins in the rat that preferentially binds heme 总被引:4,自引:0,他引:4
A low molecular weight protein purified from rat liver cytosol was observed to bind heme with an affinity higher than that for other organic anions. Purification was achieved by two procedures, one employing affinity chromatography on oleic acid-agarose, and the other using sequential ion-exchange and gel filtration chromatography after initial removal of aprotinin-sensitive proteases. Removal rather than inhibition of proteases improved the yield four times. Both procedures produced a stable protein. The purified protein binds heme with a higher affinity (Kd 0.15 microM) than any other organic anion tested including other (metallo)porphyrins, bilirubin, and oleic acid. Based on its molecular weight, amino acid composition, immunological properties, and the increase of its tissue levels in response to the administration of hypolipidemic agents, the protein was identified as being related to proteins of the Z class, whose members include fatty acid binding protein and sterol carrier protein. Like other Z proteins, our protein exhibits several forms on electrofocusing, but differs from fatty acid-binding protein and sterol carrier protein in that its major form exhibits a pI of 7.4. In view of its distinct isoelectric focusing pattern, its higher affinity for heme than for oleic acid, and its apparent inability to bind cholesterol and steroids, we cannot identify this protein as any of the above-mentioned proteins of the Z class. Consequently we have provisionally designated it heme-binding protein. 相似文献
18.
E Krzywańska-Pajdak 《Acta microbiologica Polonica》1983,32(2):131-138
Two extracellular proteases have been isolated from the culture supernatant of a virulent strain of Pseudomonas aeruginosa. The enzymes were purified in a three-step procedure involving ammonium sulfate fractionation, acetone precipitation and column chromatography on DE-52 cellulose. The specific activity of protease I was 22.2 U/mg of protein and protease II 6.6 U/mg of protein. Immunological properties and electrophoretic mobilities of the two forms were different. The two forms differ in substrate specificity (only from I exhibited elastinolytic activity) and pH optimum (pH 7.5 and pH 10 for form I and II, respectively). 相似文献
19.
Studies of Streptomyces reticuli cel-1 (cellulase) gene expression in Streptomyces strains, Escherichia coli, and Bacillus subtilis. 总被引:1,自引:0,他引:1
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Various streptomyces strains [Streptomyces lividans 66, Streptomyces vinaceus, and Strepotmyces coelicolor A3 (2)] acquired the ability to utilize crystalline cellulose (Avicel) after transformation with a multicopy vector containing the cel-1 gene from Streptomyces reticuli. The expression level in these hosts was two to three times lower than in S. reticuli, indicating the absence of positive regulatory elements. Like S. reticuli, they processed the Avicelase to its catalytic domain and to an enzymatically inactive part. The cel-1 gene with its original upstream region was not expressed within Escherichia coli. When cel-1 had been fused in phase with the lacZ gene, large quantities of the fusion protein were produced in E. coli. However, this protein was enzymatically inactive and proteolytically degraded to a series of truncated forms. As the cellulase (Avicelase) synthesized by S. reticuli is not cleaved by the E. coli proteases, its posttranslational modification is proposed. With Bacillus subtilis as host, the cel-1 gene was expressed neither under its own promoter nor under the control of a strong Bacillus promoter. 相似文献