Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).
ABSTRACT: BACKGROUND:Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. RESULTS:In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm's excretory-secretory products (ESPs) was confirmed. CONCLUSION:EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin's presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.
Project description:Inhibitory serpins are metastable proteins that undergo a substantial conformational rearrangement to covalently trap target peptidases. The serpin reactive center loop contributes a majority of the interactions that serpins make during the initial binding to target peptidases. However, structural studies on serpin-peptidase complexes reveal a broader set of contacts on the scaffold of inhibitory serpins that have substantial influence on guiding peptidase recognition. Structural and biophysical studies also reveal how aberrant serpin folding can lead to the formation of domain-swapped serpin multimers rather than the monomeric metastable state. Serpin domain swapping may therefore underlie the polymerization events characteristic of the serpinopathies. Finally, recent structural studies reveal how the serpin fold has been adapted for non-inhibitory functions such as hormone binding.
Project description:The serpins (serine proteinase inhibitors) are structurally similar but functionally diverse proteins that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. Serpins play absolutely critical role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways and are associated with many conformational diseases. Serpin's native state is a metastable state which transforms to a more stable state during its inhibitory mechanism. Serpin in the native form is in the stressed (S) conformation that undergoes a transition to a relaxed (R) conformation for the protease inhibition. During this transition the region called as reactive center loop which interacts with target proteases, inserts itself into the center of ?-sheet A to form an extra strand. Serpin is delicately balanced to perform its function with many critical residues involved in maintaining metastability. However due to its typical mechanism of inhibition, naturally occurring serpin variants produces conformational instability that allows insertion of RCL of one molecule into the ?-sheet A of another to form a loop-sheet linkage leading to its polymerization and aggregation. Thus understanding the molecular basis and amino acid involved in serpin polymerization mechanism is critical to devising strategies for its cure.
Project description:The clade B/intracellular serpins protect cells from peptidase-mediated injury by forming covalent complexes with their targets. SERPINB12 is expressed in most tissues, especially at cellular interfaces with the external environment. This wide tissue distribution pattern is similar to that of granzyme A (GZMA). Because SERPINB12 inhibits trypsin-like serine peptidases, we determined whether it might also neutralize GZMA. SERPINB12 formed a covalent complex with GZMA and inhibited the enzyme with typical serpin slow-binding kinetics. SERPINB12 also inhibited Hepsin. SERPINB12 may function as an endogenous inhibitor of these peptidases.
Project description:Extracellular serpins such as antithrombin and alpha1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.
Project description:BACKGROUND: Most members of the serpin family of proteins are potent, irreversible inhibitors of specific serine or cysteine proteinases. Inhibitory serpins are distinguished from members of other families of proteinase inhibitors by their metastable structure and unique suicide-substrate mechanism. Animal serpins exert control over a remarkable diversity of physiological processes including blood coagulation, fibrinolysis, innate immunity and aspects of development. Relatively little is known about the complement of serpin genes in plant genomes and the biological functions of plant serpins. RESULTS: A structurally refined amino-acid sequence alignment of the 14 full-length serpins encoded in the genome of the japonica rice Oryza sativa cv. Nipponbare (a monocot) showed a diversity of reactive-centre sequences (which largely determine inhibitory specificity) and a low degree of identity with those of serpins in Arabidopsis (a eudicot). A new convenient and functionally informative nomenclature for plant serpins in which the reactive-centre sequence is incorporated into the serpin name was developed and applied to the rice serpins. A phylogenetic analysis of the rice serpins provided evidence for two main clades and a number of relatively recent gene duplications. Transcriptional analysis showed vastly different levels of basal expression among eight selected rice serpin genes in callus tissue, during seedling development, among vegetative tissues of mature plants and throughout seed development. The gene OsSRP-LRS (Os03g41419), encoding a putative orthologue of Arabidopsis AtSerpin1 (At1g47710), was expressed ubiquitously and at high levels. The second most highly expressed serpin gene was OsSRP-PLP (Os11g11500), encoding a non-inhibitory serpin with a surprisingly well-conserved reactive-centre loop (RCL) sequence among putative orthologues in other grass species. CONCLUSIONS: The diversity of reactive-centre sequences among the putatively inhibitory serpins of rice point to a range of target proteases with different proteolytic specificities. Large differences in basal expression levels of the eight selected rice serpin genes during development further suggest a range of functions in regulation and in plant defence for the corresponding proteins.
Project description:UNLABELLED:Serine Protease inhibitors (Serpins) like antithrombin, antitrypsin, neuroserpin, antichymotrypsin, protein C-inhibitor and plasminogen activator inhibitor is involved in important biological functions like blood coagulation, fibrinolysis, inflammation, cell migration and complement activation. Serpins native state is metastable, which undergoes transformation to a more stable state during the process of protease inhibition. Serpins are prone to conformation defects, however little is known about the factors and mechanisms which promote its conformational change and misfolding. Helix B region in serpins is with several point mutations which result in pathological conditions due to polymerization. Helix B analysis for residue burial and cavity was undertaken to understand its role in serpin structure function. A structural overlap and an accessible surface area analysis showed the deformation of strand 6B and exposure of helix B at N-terminal end in cleaved conformation but not in the native and latent conformation of various inhibitory serpins. A cleaved polymer like conformation of antitrypsin also showed deformation of s6B and helix B exposure. Cavity analysis showed that helix B residues were part of the largest cavity in most of the serpins in the native state which increase in size during the transformation to cleaved and latent states. These data for the first time show the importance of strand 6B deformation and exposure of helix B in smooth insertion of the reactive center loop during serpin inhibition and indicate that helix B exposure due to variants may increase its polymer propensity. ABBREVIATIONS:serpin -serine protease inhibitors RCL -reactive center loop ASA -accessible surface area.
Project description:The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest ?-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates.
Project description:Serpins are a widely distributed family of high molecular mass protein proteinase inhibitors that can inhibit both serine and cysteine proteinases by a remarkable mechanism-based kinetic trapping of an acyl or thioacyl enzyme intermediate that involves massive conformational transformation. The trapping is based on distortion of the proteinase in the complex, with energy derived from the unique metastability of the active serpin. Serpins are the favoured inhibitors for regulation of proteinases in complex proteolytic cascades, such as are involved in blood coagulation, fibrinolysis and complement activation, by virtue of the ability to modulate their specificity and reactivity. Given their prominence as inhibitors, much work has been carried out to understand not only the mechanism of inhibition, but how it is fine-tuned, both spatially and temporally. The metastability of the active state raises the question of how serpins fold, whereas the misfolding of some serpin variants that leads to polymerization and pathologies of liver disease, emphysema and dementia makes it clinically important to understand how such polymerization might occur. Finally, since binding of serpins and their proteinase complexes, particularly plasminogen activator inhibitor-1 (PAI-1), to the clearance and signalling receptor LRP1 (low density lipoprotein receptor-related protein 1), may affect pathways linked to cell migration, angiogenesis, and tumour progression, it is important to understand the nature and specificity of binding. The current state of understanding of these areas is addressed here.
Project description:BACKGROUND: Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control. METHODS: SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes. RESULTS: The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1' was translocated to the putative position P4'-P5'. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine ?-helices and three ?-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus. CONCLUSIONS: For the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule.
Project description:Serpins form an enormous superfamily of 40-60-kDa proteins found in almost all types of organisms, including humans. Most are one-use suicide substrate serine and cysteine proteinase inhibitors that have evolved to finely regulate complex proteolytic pathways, such as blood coagulation, fibrinolysis, and inflammation. Despite distinct functions for each serpin, there is much redundancy in the primary specificity-determining residues. However, many serpins exploit additional exosites to generate the exquisite specificity that makes a given serpin effective only when certain other criteria, such as the presence of specific cofactors, are met. With a focus on human serpins, this minireview examines use of exosites by nine serpins in the initial complex-forming phase to modulate primary specificity in either binary serpin-proteinase complexes or ternary complexes that additionally employ a protein or other cofactor. A frequent theme is down-regulation of inhibitory activity unless the exosite(s) are engaged. In addition, the use of exosites by maspin and plasminogen activator inhibitor-1 to indirectly affect proteolytic processes is considered.