Myroilysin Is a New Bacterial Member of the M12A Family of Metzincin Metallopeptidases and Is Activated by a Cysteine Switch Mechanism.
ABSTRACT: Proteases play important roles in all living organisms and also have important industrial applications. Family M12A metalloproteases, mainly found throughout the animal kingdom, belong to the metzincin protease family and are synthesized as inactive precursors. So far, only flavastacin and myroilysin, isolated from bacteria, were reported to be M12A proteases, whereas the classification of myroilysin is still unclear due to the lack of structural information. Here, we report the crystal structures of pro-myroilysin from bacterium Myroides sp. cslb8. The catalytic zinc ion of pro-myroilysin, at the bottom of a deep active site, is coordinated by three histidine residues in the conserved motif HEXXHXXGXXH; the cysteine residue in the pro-peptide coordinates the catalytic zinc ion and inhibits myroilysin activity. Structure comparisons revealed that myroilysin shares high similarity with the members of the M12A, M10A, and M10B families of metalloproteases. However, a unique "cap" structure tops the active site cleft in the structure of pro-myroilysin, and this "cap" structure does not exist in the above structure-reported subfamilies. Further structure-based sequence analysis revealed that myroilysin appears to belong to the M12A family, but pro-myroilysin uses a "cysteine switch" activation mechanism with a unique segment, including the conserved cysteine residue, whereas other reported M12A family proteases use an "aspartate switch" activation mechanism. Thus, our results suggest that myroilysin is a new bacterial member of the M12A family with an exceptional cysteine switch activation mechanism. Our results shed new light on the classification of the M12A family and may suggest a divergent evolution of the M12 family.
Project description:Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases.
Project description:The full-length sequence of tick salivary gland cDNA coding for a protein similar to metalloproteases (MP) of the reprolysin family is reported. The Ixodes scapularis MP is a 488 amino acid (aa) protein containing pre- and pro-enzyme domains, the zinc-binding motif HExxHxxGxxH common to metalloproteases, and a cysteine-rich region. In addition, the predicted amino-terminal sequences of I. scapularis MPs were found by Edman degradation of PVDF-transferred SDS/PAGE-separated tick saliva proteins, indicating that these putative enzymes are secreted. Furthermore, saliva has a metal-dependent proteolytic activity towards gelatin, fibrin(ogen), and fibronectin, but not collagen or laminin. Accordingly, I. scapularis saliva has a rather specific metalloprotease similar to the hemorrhagic proteases of snake venoms. This is the first description of such activity in tick saliva and its role in tick feeding and Borrelia transmission is discussed.
Project description:The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.
Project description:The helper-component proteinase (HC-Pro) of potyvirus is involved in polyprotein processing, aphid transmission, and suppression of antiviral RNA silencing. There is no high resolution structure reported for any part of HC-Pro, hindering mechanistic understanding of its multiple functions. We have determined the crystal structure of the cysteine protease domain of HC-Pro from turnip mosaic virus at 2.0 Å resolution. As a protease, HC-Pro only cleaves a Gly-Gly dipeptide at its own C terminus. The structure represents a postcleavage state in which the cleaved C terminus remains tightly bound at the active site cleft to prevent trans activity. The structure adopts a compact α/β-fold, which differs from papain-like cysteine proteases and shows weak similarity to nsP2 protease from Venezuelan equine encephalitis alphavirus. Nevertheless, the catalytic cysteine and histidine residues constitute an active site that is highly similar to these in papain-like and nsP2 proteases. HC-Pro recognizes a consensus sequence YXVGG around the cleavage site between the two glycine residues. The structure delineates the sequence specificity at sites P1-P4. Structural modeling and covariation analysis across the Potyviridae family suggest a tryptophan residue accounting for the glycine specificity at site P1'. Moreover, a surface of the protease domain is conserved in potyvirus but not in other genera of the Potyviridae family, likely due to extra functional constrain. The structure provides insight into the catalysis mechanism, cis-acting mode, cleavage site specificity, and other functions of the HC-Pro protease domain.
Project description:Like most metalloproteases, matrix metalloprotease 2 (MMP-2) is synthesized as a zymogen. MMP-2 propeptide plays a role in inhibition of catalytic activity through a cysteine-zinc ion pairing, disruption of which results in full enzyme activation. A variety of proteases have been shown to be involved in the activation of pro-MMP-2, including metalloproteases and serine proteases. In the previous study we showed that MMP-2 activation occurred via specific cleavages of the propeptide by thrombin followed by intermolecular autoproteolytic processing for full enzymatic activity. Thrombin also degraded MMP-2, but this degradation was reduced greatly under cell-associated conditions with a concomitant increase in activation, prompting us to elucidate the molecular mechanisms underlying thrombin-mediated MMP-2 activation. In the present study we demonstrate that heparan sulfate is essential for thrombin-mediated activation of pro-MMP-2. Binding of heparan sulfate to thrombin is primarily responsible for this activation process, presumably through conformational changes at the active site. Furthermore, interaction of MMP-2 with exosites 1 and 2 of thrombin is crucial for thrombin-mediated MMP-2 degradation, and inhibition of this interaction by heparan sulfate or hirudin fragment results in a decrease in MMP-2 degradation. Finally, we demonstrated interaction between exosite 1 and hemopexin-like domain of MMP-2, suggesting a regulatory role of hemopexin-like domain in MMP-2 degradation. Taken together, our experimental data suggest a novel regulatory mechanism of thrombin-dependent MMP-2 enzymatic activity by heparan sulfate proteoglycans.
Project description:BACKGROUND:Naegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. Cysteine proteases produced by the amoeba may play critical roles in the pathogenesis of infection. In this study, a novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was characterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells. METHODS:Recombinant fowlerstefin was expressed in Escherichia coli. The inhibitory activity of fowlerstefin against several cysteine proteases, including human cathepsins B and L, papain and NfCPB-L, was analyzed. Fowlerstefin-induced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS:Fowlerstefin is a cysteine protease inhibitor with a monomeric structure, and belongs to the stefin family. Recombinant fowlerstefin effectively inhibited diverse cysteine proteases including cathepsin B-like cysteine proteases of N. fowleri (NfCPB-L), human cathepsins B and L, and papain. Expression of fowlerstefin in the amoeba was optimal during the trophozoite stage and gradually decreased in cysts. Fowlerstefin induced an inflammatory response in BV-2 microglial cells. Fowlerstefin induced the expression of several pro-inflammatory cytokines and chemokines including IL-6 and TNF in BV-2 microglial cells. Fowlerstefin-induced expression of IL-6 and TNF in BV-2 microglial cells was regulated by mitogen-activated protein kinase (MAPKs). The inflammatory response induced by fowlerstefin in BV-2 microglial cells was downregulated via inhibition of NF-?B and AP-1. CONCLUSIONS:Fowlerstefin is a pathogenic molecule that stimulates BV-2 microglial cells to produce pro-inflammatory cytokines through NF-?B- and AP-1-dependent MAPK signaling pathways. Fowlerstefin-induced inflammatory cytokines exacerbate the inflammatory response in N. fowleri-infected areas and contribute to the pathogenesis of PAM.
Project description:Nearly all high-molecular-weight (HMW) dissolved organic nitrogen and part of the particulate organic nitrogen in the deep sea are present in hydrolysis-resistant amides, and so far the mechanisms of biodegradation of these types of nitrogen have not been resolved. The M12 family is the second largest family in subclan MA(M) of Zn-containing metalloproteases and includes most enzymes from animals and only one enzyme (flavastacin) from a human-pathogenic bacterium (Flavobacterium meningosepticum). Here, we characterized the novel M12 protease myroilysin with elastinolytic activity and collagen-swelling ability from the newly described deep-sea bacterium Myroides profundi D25. Myroilysin is a monomer enzyme with 205 amino acid residues and a molecular mass of 22,936 Da. It has the same conserved residues at the four zinc ligands as astacin and very low levels of identity (<or=40%) to other metalloproteases, indicating that it is a novel metalloprotease belonging to subfamily M12A. Myroilysin had broad specificity and much higher elastinolytic activity than the bacterial elastinase pseudolysin. To our knowledge, it is the first reported elastase in the M12 family. Although it displayed very low activity with collagen, myroilysin had strong collagen-swelling ability and played a synergistic role with collagenase in collagen hydrolysis. It can be speculated that myroilysin synergistically interacts with other enzymes in its in situ biotic assemblage and that it may play an important role in the degradation of deep-sea HMW organic nitrogen.
Project description:Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (N(pro)) is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, N(pro) is no longer active as a protease. The released N(pro) suppresses the induction of the host's type-I interferon-α/β (IFN-α/β) response. N(pro) binds interferon regulatory factor-3 (IRF3), the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus N(pro). N(pro) is structurally distinct from other known cysteine proteases and has a novel "clam shell" fold consisting of a protease domain and a zinc-binding domain. The unique fold of N(pro) allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of N(pro) involved in targeting IRF3 for proteasomal degradation provides insight into the nature of N(pro)'s interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of N(pro), and its role in subversion of host immune response.
Project description:Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.
Project description:Ochroconis mirabilis, a recently introduced water-borne dematiaceous fungus, is occasionally isolated from human skin lesions and nails. We identified an isolate of O. mirabilis from a skin scraping with morphological and molecular studies. Its genome was then sequenced and analysed for genetic features related to classification and biological characteristics.UM 578 was identified as O. mirabilis based on morphology and internal transcribed spacer (ITS)-based phylogeny. The 34.61 Mb assembled genome with 13,435 predicted genes showed less efficiency of this isolate in plant cell wall degradation. Results from the peptidase comparison analysis with reported keratin-degrading peptidases from dermatophytes suggest that UM 578 is very unlikely to be utilising these peptidases to survive in the host. Nevertheless, we have identified peptidases from M10A, M12A and S33 families that may allow UM 578 to invade its host via extracellular matrix and collagen degradation. Furthermore, the lipases in UM 578 may have a role in supporting the fungus in host invasion. This fungus has the potential ability to synthesise melanin via the 1,8-dihydroxynaphthalene (DHN)-melanin pathway and to produce mycotoxins. The mating ability of this fungus was also inspected in this study and a mating type gene containing alpha domain was identified. This fungus is likely to produce taurine that is required in osmoregulation. The expanded gene family encoding the taurine catabolism dioxygenase TauD/TdfA domain suggests the utilisation of taurine under sulfate starvation. The expanded glutathione-S-transferase domains and RTA1-like protein families indicate the selection of genes in UM 578 towards adaptation in hostile environments.The genomic analysis of O. mirabilis UM 578 provides a better understanding of fungal survival tactics in different habitats.