Identification, distribution and molecular evolution of the pacifastin gene family in Metazoa

Background  

Members of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins. Structural and biochemical characteristics of insect pacifastin-like peptides have been studied intensively, but only one inhibitor has been functionally characterised. Recent sequencing projects of metazoan genomes have created an unprecedented opportunity to explore the distribution, evolution and functional diversification of pacifastin genes in the animal kingdom.     

A simple fold with variations: the pacifastin inhibitor family

Members of the pacifastin family are small, approximately 35-residue serine protease inhibitors isolated from arthropod species. Several locust inhibitors exhibit intriguing taxon specificity while others do not. The structural basis of this phenomenon may lie in the different dynamical properties of the proteins originating from different stabilizing interactions. In this study, we identify new members of the family to confirm the universal role of these interactions in the family. Structural investigations show that both the disulfide pattern and the stabilizing interactions are unique among small all-beta proteins.     

In vitro activity of pacifastin-like inhibitors in relation to their structural characteristics

Information on the structural characteristics and inhibitory activity of the pacifastin family is restricted to a handful of locust pacifastin-related inhibitors. In this report the optimization of a bacterial recombinant expression system is described, resulting in the high yield production of pacifastin-like inhibitors of the desert locust. Subsequently, the relative inhibitory activity of these peptides towards mammalian, locust and caterpillar digestive peptidases has been compared. In general, the enzyme specificity of locust pacifastin-like inhibitors towards trypsin- or chymotrypsin-like peptidases corresponds to the nature of the P1-residue at the reactive site. In addition, other structural characteristics, including specific core interactions, have been reported to result in a different affinity of pacifastin members towards digestive trypsin-like enzymes from mammals and arthropods. One remarkable observation in this study is a specifically designed pacifastin-like peptidase inhibitor, which, unlike other inhibitors of the same family, does not display this specificity and selectivity towards digestive enzymes from different animals.     

Selective inhibition of trypsins by insect peptides: role of P6-P10 loop

PMP-D2 and HI, two peptides from Locusta migratoria, were shown to belong to the family of tight-binding protease inhibitors. However, they interact weakly with bovine trypsin (K(i) around 100 nM) despite a trypsin-specific Arg at the primary specificity site P1. Here we demonstrate that they are potent inhibitors of midgut trypsins isolated from the same insect and of a fungal trypsin from Fusarium oxysporum (K(i) 相似文献   

Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition but, due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be aided greatly by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.     

Design and synthesis of a series of serine derivatives as small molecule inhibitors of the SARS coronavirus 3CL protease

Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai’s cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity.     

Structure-activity relationship within the serine protease inhibitors of the pacifastin family

The members of the Pacifastin family are serine protease inhibitors found in insects and crustacean. They are either small inhibitors (made of one consensus cysteine-rich motif) or proteins (4-9 motifs). Some of these inhibitors are characterized by a species selectivity for the trypsin inhibition. Structural data discriminate the small inhibitors that apparently look very similar into two groups. Interestingly, the inhibitors that display species selectivity fall in the same structural group.     

Structural and functional properties of a novel serine protease inhibiting peptide family in arthropods

Recently, several arthropod peptides that belong to a new serine protease inhibitor family were discovered. Three members (HI, PMP-D2=LMCI-1 and PMP-C=LMCI-2) were isolated from the migratory locust, Locusta migratoria. Five additional members (SGPI-1-5) were identified in the desert locust Schistocerca gregaria, and a heterodimeric serine protease inhibitor (pacifastin) was isolated from the hemolymph of the crayfish Pacifastacus leniusculus. The light chain of pacifastin constitutes the inhibitory subunit that has nine cysteine-rich domains (PLDs) that are homologous with the locust inhibitors. These locust inhibitors and PLDs share a conserved array of six cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys), which are involved in an identical disulfide bridge pattern (Cys(1)-Cys(4), Cys(2)-Cys(6), Cys(3)-Cys(5)). The solution structures of LMCI-1 and LMCI-2 showed a similar, compact, globular folding, which is unique within the group of the small 'canonical' inhibitors. Moreover, the reactive site, including the P1-P'1 bond was thoroughly investigated by means of synthetic variants. However, the biological function(s) of the locust inhibitors is (are) not fully understood. LMCI-1 and LMCI-2 were shown to inhibit the endogenous proteolytic activating cascade of prophenoloxidase. Northern blot analysis indicated that the genes encoding the SGPI precursors are differentially expressed in a time-, stage- and hormone-dependent manner.     

Local binding with globally distributed changes in a small protease inhibitor upon enzyme binding

Complexation of the small serine protease inhibitor Schistocerca gregaria chymotrypsin inhibitor (SGCI), a member of the pacifastin inhibitor family, with bovine chymotrypsin was followed by NMR spectroscopy. (1)H-(15)N correlation (HSQC) spectra of the inhibitor with increasing amounts of the enzyme reveal tight and specific binding in agreement with biochemical data. Unexpectedly, and unparalleled among canonical serine protease inhibitors, not only residues in the protease-binding loop of the inhibitor, but also some segments of it located spatially far from the substrate-binding cleft of the enzyme were affected by complexation. However, besides changes, some of the dynamical features of the free inhibitor are retained in the complex. Comparison of the free and complexed inhibitor structures revealed that most, but not all, of the observed chemical shift changes can be attributed to minor structural transitions. We suggest that the classical 'scaffold + binding loop' model of canonical inhibitors might not be fully valid for the inhibitor family studied. In our view, this feature allows for the emergence of both taxon-specific and nontaxon-specific inhibitors in this group of small proteins.     

Eisenstasin,new antistasin family inhibitor from the earthworm

A couple of new antistasin family serine protease inhibitors have been isolated from the non-hematophagous earthworm, Eisenia andrei. These novel inhibitors have been designated as eisenstasin I and II. Similar to other antistasin family inhibitors, eisenstasin I and II feature 3 and 4 internal repeats, respectively, of a 24–29 amino acid sequence, both of which exhibit a conserved pattern of 6-cysteine/2-glycine at an identical position between the third and fourth cysteine residues. This suggests that the eisenstasins isolated from the earthworm are members of the antistasin family. The eisenstasins are 82% similar with regard to amino acid sequences and exhibit over 70% similarity with the antistasins from the earthworm Lumbricus rubellus, while also displaying less than 40% sequence similarity with the leech antistasins. Earthworm eisenstasins are basic proteins, primarily due to the frequent occurrence of arginine residues in their structure, especially at the C-terminal region. As arginine is a key residue for the substrate specificity of some serine proteases including FXa, it is thought that these multiple arginine residues may play a role in the inhibitory characteristics of the eisenstasins. Considering the structure and number of the internal repeats derived from a variety of animal species, the deletion as well as the duplication of all or part of an internal repeat may be implicated in the evolution of the structure and function of the antistasin family inhibitors.     

Genomics, evolution and biological functions of the pacifastin peptide family: a conserved serine protease inhibitor family in arthropods

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5). The light chain of the heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of six conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-6)-Cys-Thr-Xaa(3)-Cys) with the locust inhibitors. Via cDNA cloning, eight pacifastin-related precursors have been identified in locusts. Interestingly, additional pacifastin-related precursors have been identified in Diptera, Lepidoptera and Coleoptera utilising an in silico data mining approach.     

TGF-beta signaling from a three-dimensional perspective: insight into selection of partners

Members of the transforming growth factor beta (TGF-beta) family, which include TGF-beta s, activins and bone morphogenetic proteins (BMPs), are potent regulators of cell proliferation, differentiation, migration and apoptosis. They act through binding to and activating serine/threonine kinase receptors on the cell surface and triggering intracellular signaling pathways in which Smad proteins have essential roles. Here, we discuss recent structure-based studies of TGF-beta s and BMPs, their receptors, and of Smad proteins, which have unravelled insights into ligand specificity, receptor and Smad activation, as well as new features of Smads as phosphoserine-binding entities.     

Identification of novel protein kinase CK1 delta (CK1delta) inhibitors through structure-based virtual screening

In eukaryotes, protein phosphorylation of serine, threonine or tyrosine residues by protein kinases plays an important role in many cellular processes. Members of the protein kinase CK1 family usually phosphorylate residues of serine that are close to other phosphoserine in a consensus motif of pS-X-X-S, and they are implicated in the regulation of a variety of physiological processes as well as in pathologies like cancer and Alzheimer's disease. Using a structure-based virtual screening (SBVS) approach we have identified two anthraquinones as novel CK1delta inhibitors. These amino-anthraquinone analogs (derivatives 1 and 2) are among the most potent and selective CK1delta inhibitors known today (IC(50)=0.3 and 0.6 microM, respectively).     

Additivity-based prediction of equilibrium constants for some protein-protein associations

For many protein families, such as serine proteinases or serine proteinase inhibitors, the family assignment predicts reactivity only in general terms. Both detailed specificity and quantitative reactivity are lacking. We believe that, for many such protein families, algorithms can be devised by defining the subset of n functionally important sequence positions, making the 19n possible single mutants and measuring their reactivity. Given the assumption that the contributions of the n positions are additive, the reactivities of the 20(n) variants can be predicted. This is illustrated by an almost complete algorithm for the Kazal family of protein inhibitors of serine proteinases.     

Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

For functional studies, nine cDNAs encoding Kunitz-type enzyme inhibitors from potato tubers were expressed as GST (glutathione S transferase)-tagged fusion proteins in the fission yeast Schizosaccharomyces pombe. The inhibitors represented the three major homology groups A, B and C found in tubers. Members of the same homology group were at least 90% identical in sequence. The purified GST fusion proteins were tested for their ability to inhibit the proteases trypsin, alpha-chymotrypsin, subtilisin, papain and aspergillopepsin I, and for inhibition of the growth of fungi. Fusion proteins belonging to the same and different homology groups were found to exhibit distinct protease inhibition profiles. Removal of the GST tag by cleavage with enterokinase did not change the inhibition profile but increased the inhibitory activity. Group A and B inhibitors affected the proteases to different extents, whereas group C inhibitors showed only weak or no protease inhibition. One fusion protein completely inhibited aspergillopepsin I. One fusion protein each of groups A and B strongly inhibited mycelial growth of the fungus Fusarium moniliforme. The results suggest functional polymorphism among closely related members of the Kunitz-type inhibitor family.     

Evolution of Bacillus thuringiensis Cry toxins insecticidal activity

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate‐limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.     

Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function

In metazoan organisms protein inhibitors of peptidases are important factors essential for regulation of proteolytic activity. In vertebrates genes encoding peptidase inhibitors constitute up to 1% of genes reflecting a need for tight and specific control of proteolysis especially in extracellular body fluids. In stark contrast unicellular organisms, both prokaryotic and eukaryotic consistently contain only few, if any, genes coding for putative peptidase inhibitors. This may seem perplexing in the light of the fact that these organisms produce large numbers of proteases of different catalytic classes with the genes constituting up to 6% of the total gene count with the average being about 3%. Apparently, however, a unicellular life-style is fully compatible with other mechanisms of regulation of proteolysis and does not require protein inhibitors to control their intracellular and extracellular proteolytic activity. So in prokaryotes occurrence of genes encoding different types of peptidase inhibitors is infrequent and often scattered among phylogenetically distinct orders or even phyla of microbiota. Genes encoding proteins homologous to alpha-2-macroglobulin (family I39), serine carboxypeptidase Y inhibitor (family I51), alpha-1-peptidase inhibitor (family I4) and ecotin (family I11) are the most frequently represented in Bacteria. Although several of these gene products were shown to possess inhibitory activity, with an exception of ecotin and staphostatins, the biological function of microbial inhibitors is unclear. In this review we present distribution of protein inhibitors from different families among prokaryotes, describe their mode of action and hypothesize on their role in microbial physiology and interactions with hosts and environment.     

In-cell selectivity profiling of serine protease inhibitors by activity-based proteomics

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.     

Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.