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In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease composed of ClpY (HslU), an ATPase with unfolding activity, and ClpQ (HslV), a peptidase. In the ClpYQ proteolytic complex, the hexameric rings of ClpY (HslU) are responsible for protein recognition, unfolding, and translocation into the proteolytic inner chamber of the dodecameric ClpQ (HslV). Each of the three domains, N, I, and C, in ClpY has its own distinct activity. The double loops (amino acids [aa] 137 to 150 and 175 to 209) in domain I of ClpY are necessary for initial recognition/tethering of natural substrates such as SulA, a cell division inhibitor protein. The highly conserved sequence GYVG (aa 90 to 93) pore I site, along with the GESSG pore II site (aa 265 to 269), contribute to the central pore of ClpY in domain N. These two central loops of ClpY are in the center of its hexameric ring in which the energy of ATP hydrolysis allows substrate translocation and then degradation by ClpQ. However, no data have been obtained to determine the effect of the central loops on substrate binding or as part of the processivity of the ClpYQ complex. Thus, we probed the features of ClpY important for substrate engagement and protease processivity via random PCR or site-specific mutagenesis. In yeast two-hybrid analysis and pulldown assays, using isolated ClpY mutants and the pore I or pore II site of ClpY, each was examined for its influence on the adjoining structural regions of the substrates. The pore I site is essential for the translocation of the engaged substrates. Our in vivo study of the ClpY mutants also revealed that an ATP-binding site in domain N, separate from its role in polypeptide (ClpY) oligomerization, is required for complex formation with ClpQ. Additionally, we found that the tyrosine residue at position 408 in ClpY is critical for stabilization of hexamer formation between subunits. Therefore, our studies suggest that stepwise activities of the ClpYQ protease are necessary to facilitate the processive degradation of its natural substrates.  相似文献   

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The ATP-dependent ClpQY protease system in Plasmodium falciparum is a prokaryotic machinery in the parasite. In the present study, we have identified the complete ClpQY system in P. falciparum and elucidated its functional importance in survival and growth of asexual stage parasites. We characterized the interaction of P. falciparum ClpQ protease (PfClpQ) and PfClpY ATPase components, and showed that a short stretch of residues at the C terminus of PfClpY has an important role in this interaction; a synthetic peptide corresponding to this region antagonizes this interaction and interferes with the functioning of this machinery in the parasite. Disruption of ClpQY function by this peptide caused hindrance in the parasite growth and maturation of asexual stages of parasites. Detailed analyses of cellular effects in these parasites showed features of apoptosis-like cell death. The peptide-treated parasites showed mitochondrial dysfunction and loss of mitochondrial membrane potential. Dysfunctioning of mitochondria initiated a cascade of reactions in parasites, including activation of VAD–FMK-binding proteases and nucleases, which resulted in apoptosis-like cell death. These results show functional importance of mitochondrial proteases in the parasite and involvement of mitochondria in programmed cell death in the malaria parasites.  相似文献   

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Proteolysis constitutes a major post-translational modification but specificity and substrate selectivity of numerous proteases have remained elusive. In this review, we highlight how advanced techniques in the areas of proteomics and activity-based probes can be used to investigate i) protease active site specificity; ii) protease in vivo substrates; iii) protease contribution to proteome homeostasis and composition; and iv) detection and localization of active proteases. Peptide libraries together with genetical or biochemical selection have traditionally been used for active site profiling of proteases. These are now complemented by proteome-derived peptide libraries that simultaneously determine prime and non-prime specificity and characterize subsite cooperativity. Cell-contextual discovery of protease substrates is rendered possible by techniques that isolate and quantitate protein termini. Here, a novel approach termed Terminal Amine Isotopic Labeling of Substrates (TAILS) provides an integrated platform for substrate discovery and appropriate statistical evaluation of terminal peptide identification and quantification. Proteolytically generated carboxy-termini can now also be analyzed on a proteome-wide level. Proteolytic regulation of proteome composition is monitored by quantitative proteomic approaches employing stable isotope coding or label free quantification. Activity-based probes specifically recognize active proteases. In proteomic screens, they can be used to detect and quantitate proteolytic activity while their application in cellular histology allows to locate proteolytic activity in situ. Activity-based probes – especially in conjunction with positron emission tomography – are also promising tools to monitor proteolytic activities on an organism-wide basis with a focus on in vivo tumor imaging. Together, this array of methodological possibilities enables unveiling physiological protease substrate repertoires and defining protease function in the cellular- and organism-wide context.  相似文献   

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Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.  相似文献   

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Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.  相似文献   

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Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.  相似文献   

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《Process Biochemistry》2010,45(1):133-137
The nucleocapsid (N) protein of Nipah virus (NiV) expressed in Escherichia coli (E. coli) is antigenic and immunogenic. A method to enhance the recovery of recombinant N protein of NiV produced in E. coli is described. A bioinformatics tool, PeptideCutter was used to identify potential protease and cleavage sites from the amino acid sequences deduced from the published DNA sequence of the N protein of NiV. The size of degraded protein was estimated by using the Western blot and PeptideCutter analyse. The identified proteases were serine proteases, hence, a range of serine protease inhibitors were tested to improve the recovery of the N protein. The relative amount of N protein of NiV was 2-fold higher with the addition of PMSF, compared to the control sample (without any protease inhibitor supplementation).  相似文献   

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The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg15-Ala16 reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.  相似文献   

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Su W  Lin C  Wu J  Li K  He G  Qian X  Wei C  Yang J 《Biotechnology letters》2006,28(12):923-927
The ATP-dependent Lon protease is a highly conserved enzyme that is present in archeae, eubacteria, and eukaryotes, and plays an important role in intracellular protein degradation. We have isolated a Lon protease gene, OsLon1, from Oryza sativa. The cDNA contained a 2,655 bp ORF. Comparative analysis showed that OsLon1 shared significant similarity with the previously reported Lon proteases from maize, Arabidopsis, human, and bacteria. Tissue expression pattern analysis revealed that OsLon1 was highly expressed in young leaves, mature leaves, and leaf sheaths but only weakly in young roots, mature roots, and young panicles. The OsLon1 gene was successfully expressed in E. coli and the detected protein size, about 120 kDa, matched the expected molecular mass of the His-tagged OsLon1 protein.  相似文献   

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Background  

Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind.  相似文献   

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Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis.  相似文献   

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Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.  相似文献   

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Philasterides dicentrarchi is a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus, aquaculture in Korea. In this study, a cDNA encoding a cathepsin L-like cysteine protease (PdCtL) of P. dicentrarchi (synonym Miamiensis avidus) was identified. To express the PdCtL recombinant protein in a heterologous system, 10 codons were redesigned to conform to the standard eukaryotic genetic code using polymerase chain reaction (PCR)-based site-directed mutagenesis. The recombinant P. dicentrarchi procathepsin L (proPdCtL) was expressed at high levels in E. coli Rosetta (DE3) pLysS with a pPET21a vector, and successfully refolded, purified, and activated into a functional and enzymatically active form. The optimal pH for protease activity was 5. Similar to other cysteine proteases, enzyme activity was inhibited by E64 and leupeptin. Immunogenicity of recombinant PdCtL was assessed by enzyme-linked immunosorbent assay, western blot, and specific anti-recombinant PdCtL antibodies were detected. Our results suggest that the biochemical characteristics of the recombinant ciliate proPdCtL protein are similar to those of the cathepsin L-like cysteine protease, that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form, and that the recombinant PdCtL acted as a specific epitope in olive flounder.  相似文献   

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Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperone rings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation. Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Mycobacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2, and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently, this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobic chaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clp proteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core, and it frees the ClpP1 interaction interface for putative new binding partners.  相似文献   

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A membrane-based high-throughput screening (HTS) assay for active d-amino acid oxidase (DAAO) in liquid samples as well as in intact Escherichia coli cells has been developed and optimized. The detection limit of the assay was less than 1 ng per sample. The method proposed can be used for quantitative DAAO determination in the range of 0.13 to 3.60 ng enzyme per probe. The protocol was successfully tested to screen a library of E. coli clones containing mutant DAAOs active toward target substrates.  相似文献   

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