Developing novel anthelmintics from plant cysteine proteinases.
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ABSTRACT:
SUBMITTER: Behnke JM
PROVIDER: S-EPMC2559997 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundPlant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The nematode cuticle is composed of collagens and cuticlins, but the specific molecular target(s) for the proteinases have yet to be identified.MethodsThis study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murine GI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms.ResultsImmunohistochemistry indicated that DPY-7 collagen is a target for CPs on the cuticle of C. elegans. The time course of loss of DPY-7 from the cuticle allowed us to use it to visualise the process of cuticle disruption. There was a marked difference in the time course of damage to the cuticles of the two species of nematode, with H. bakeri being more rapidly hydrolysed. In general, the CPs' mode of attack on the nematode cuticle was by degrading the structural proteins, leading to loss of integrity of the cuticle, and finally death of the nematode. Proteomic analysis failed conclusively to identify structural targets for CPs, but preliminary data suggested that COL-87 and CUT-19 may be important targets for the CPs, the digestion of which may contribute to cuticle disruption and death of the worm. Cuticle globin was also identified as a cuticular target. The presence of more than one target protein may slow the development of resistance against this new class of anthelmintic.ConclusionsScanning electron microscopy and immunohistochemistry allowed the process of disruption of the cuticle to be followed with time. Cuticle collagens and cuticlins are molecular targets for plant cysteine proteinases. However, the presence of tyrosine cross-links in nematode cuticle proteins seriously impeded protein identification by proteomic analyses. Multiple cuticle targets exist, probably making resistance to this new anthelmintic slow to develop.
| S-EPMC8180098 | biostudies-literature
Project description:Papain-like cysteine peptidases form a big and highly diverse superfamily of proteins involved in many important biological functions, such as protein turnover, deubiquitination, tissue remodeling, blood clotting, virulence, defense, and cell wall remodeling. High sequence and structure diversity observed within these proteins hinders their comprehensive classification as well as the identification of new representatives. Moreover, in general protein databases, many families already classified as papain like lack details regarding their mechanism of action or biological function. Here, we use transitive remote homology searches and 3D modeling to newly classify 21 families to the papain-like cysteine peptidase superfamily. We attempt to predict their biological function and provide structural characterization of 89 protein clusters defined based on sequence similarity altogether spanning 106 papain-like families. Moreover, we systematically discuss observed diversity in sequences, structures, and catalytic sites. Eventually, we expand the list of human papain-related proteins by seven representatives, including dopamine receptor-interacting protein 1 as potential deubiquitinase, and centriole duplication regulating CEP76 as retaining catalytically active peptidase-like domain. The presented results not only provide structure-based rationales to already existing peptidase databases but also may inspire further experimental research focused on peptidase-related biological processes.
| S-EPMC10318531 | biostudies-literature
Project description:BACKGROUND: Cystatins and their putative targets, the families of cysteine proteinases C1A and C13 play key roles in plants. Comparative genomic analyses are powerful tools to obtain valuable insights into the conservation and evolution of the proteinases and their proteinaceous inhibitors, and could aid to elucidate issues concerning the function of these proteins. RESULTS: We have performed an evolutionary comparative analysis of cysteine proteinases C1A and C13 and their putative inhibitors in representative species of different taxonomic groups that appeared during the evolution of the Viridiplantae. The results indicate that whereas C1A cysteine proteinases are present in all taxonomic groups, cystatins and C13 cysteine proteinases are absent in some basal groups. Moreover, gene duplication events have been associated to the increasing structural and functional complexities acquired in land plants. CONCLUSION: Comparative genomic analyses have provided us valuable insights into the conservation and evolution of the cystatin inhibitory family and their putative targets, the cysteine proteinases from families C1A and C13. Functionality of both families of proteins in plants must be the result of a coevolutionary process that might have occurred during the evolution of basal and land plants leading to a complex functional relationship among them.
| S-EPMC2474614 | biostudies-literature
Project description:After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases.
| S-EPMC1153481 | biostudies-other
Project description:We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes.
| S-EPMC4450334 | biostudies-literature
Project description:A series of novel synthetic dipeptides, containing a C-terminal glyoxal grouping (-COCHO), have been tested as inhibitors against typical members of the serine- and cysteine-proteinase families. For example, the sequences benzyloxycarbonyl (Cbz)-Pro-Phe-CHO (I) and Cbz-Phe-Ala-CHO (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and cathepsin B respectively, have been found to be potent reversible inhibitors of their respective target proteinase. Thus I was found to inhibit chymotrypsin with a Ki of approximately 0.8 microM, whereas II exhibits a Ki of approximately 80 nm against cathepsin B. These Ki values are some 10-fold and 3-fold lower than those reported for the corresponding peptide-aldehyde inhibitors of chymotrypsin and cathepsin B upon which the peptidyl-glyoxals were fashioned. Unexpectedly, the sequence Cbz-Pro-Ala-CHO, which was designed to inhibit elastase-like proteinases, exhibited no inhibitory activity towards porcine pancreatic elastase, even when used at concentrations as high as 200 microM.
| S-EPMC1134361 | biostudies-other
Project description:BACKGROUND: Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. RESULTS: Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. CONCLUSIONS: Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death associated with post-germination of the coffee grain. Expression analysis of the cysteine proteinase inhibitor genes suggests that CcCPI-1 could primarily be involved in modulating the activity of grain CP activity; while CcCPI-4 may play roles modulating grain CP activity and in the protection of the young coffee seedlings from insects and pathogens. CcCPI-2 and CcCPI-3, having lower and more widespread expression, could be more general "house-keeping" CPI genes.
| S-EPMC3311568 | biostudies-literature
Project description:We have isolated and sequenced chloroplast (chl) and cytoplasmic (cyt) cysteine tRNAs from Nicotiana rustica. Both tRNAs carry a GCA anticodon but beyond that differ considerably in their nucleotide sequences. One obvious distinction resides in the presence of N6-isopentenyladenosine (i6A) and 1-methylguanosine (m1G) at position 37 in chl and cyt tRNA(Cys) respectively. In order to study the potential suppressor activity of tRNAs(Cys) we used in vitro synthesized zein mRNA transcripts in which an internal UGA stop codon had been placed in either the tobacco rattle virus (TRV)- or tobacco mosaic virus (TMV)-specific codon context. In vitro translation was carried out in a messenger- and tRNA-dependent wheat germ extract. Both tRNA(Cys) isoacceptors stimulate read-through over the UGA stop codon, however, chl tRNA(GCA)Cys is more efficient than the cytoplasmic counterpart. The UGA in the two viral codon contexts is suppressed to about the same extent by either of the two tRNAs(Cys), whereas UGA in the beta-globin context is not recognized at all. The interaction of tRNA(GCA)Cys with UGA requires an unconventional G:A base pair in the wobble position, as postulated earlier for plant tRNA(G psi A)Tyr misreading the UAA stop codon. This is the first case that a cysteine-accepting tRNA has been characterized as a natural UGA suppressor.
| S-EPMC307430 | biostudies-other
Project description:The antigen recognition by the host immune system is a complex biochemical process that requires a battery of enzymes. Cathepsins are one of the enzyme superfamilies involved in antigen degradation. We observed the up-regulation of catfish cathepsin H and L transcripts during the early stage of Edwardsiella ictaluri infection in preliminary studies, and speculated that cathepsin H and L may play roles in infection. We identified, sequenced and characterized the complete channel catfish cathepsin H and L cDNAs, which comprised 1415 and 1639 nucleotides, respectively. The open reading frames of cathepsin H appeared to encode a protein of 326-amino acid residues, which that of cathepsin L encoded a protein of 336 amino acids. The degree of conservation of the channel catfish cathepsin H and L amino acid sequences in comparison to other species ranged from 61% to 77%, and 67% to 85%, respectively. The catalytic triad and substrate binding sites are conserved in cathepsin H and L amino acid sequences. The cathepsin L transcript was expressed in all tissues examined, while the cathepsin H was expressed in restricted tissues. These results provide important information for further exploring the roles of channel catfish cathepsins in antigen processing.
| S-EPMC7111680 | biostudies-literature
Project description:Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.
| S-EPMC2253190 | biostudies-literature