The use of benzyloxycarbonyl[125I]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues.
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ABSTRACT:
SUBMITTER: Mason RW
PROVIDER: S-EPMC1133521 | biostudies-other | 1989 Nov
REPOSITORIES: biostudies-other
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Project description:A new technique has been developed to identify active proteinases in endosomes that does not require prior isolation of organelles and extraction of the active enzymes. [125I]Iodotyrosylalanyldiazomethane was reversibly conjugated to transferrin to selectively deliver it to endosomes. The protein was conjugated to the inhibitor via a disulphide bond using N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibitor portion of the conjugate bound irreversibly to active cathepsins B and L, and subsequently the reacted enzymes were separated from the transferrin after SDS/PAGE under reducing conditions. Uptake of the protein-inhibitor conjugate and incorporation of inhibitor into cathepsins was blocked at 4 degreesC, demonstrating that the conjugate enters cells by receptor-mediated endocytosis. Furthermore, endocytosed transferrin-inhibitor conjugate could be recycled back to the extracellular medium and binding to the transferrin receptor could be blocked by native transferrin. Labelling of the enzymes was not blocked by incubating cells at 16 degreesC, consistent with the majority of the reagent being targeted to endosomes. The inhibited enzymes remained conjugated to transferrin, showing that the disulphide bond between the transferrin and inhibitor was not reduced in the endosome. Results from these studies show that endosomes contain both intermediate and late biosynthetic forms of active cathepsin B, which are indistinguishable from those found in mature lysosomes. These results indicate that the active enzymes in endosomes are not early biosynthetic forms in transit to lysosomes but most probably enter the endosome via retrograde traffic from the lysosome.
| S-EPMC1219918 | biostudies-other
Project description:Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock.
| S-EPMC2559997 | 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:Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an (125)I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to (125)I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The (125)I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.
| S-EPMC3696761 | biostudies-literature
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: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
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:The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.
| S-EPMC1135546 | biostudies-other
Project description:The oxidation of a key cysteine residue (Cys106) in the parkinsonism-associated protein DJ-1 regulates its ability to protect against oxidative stress and mitochondrial damage. Cys106 interacts with a neighboring protonated Glu18 residue, stabilizing the Cys106-SO2 (-) (sulfinic acid) form of DJ-1. To study this important post-translational modification, we previously designed several Glu18 mutations (E18N, E18D, E18Q) that alter the oxidative propensity of Cys106. However, recent results suggest these Glu18 mutations cause loss of DJ-1 dimerization, which would severely compromise the protein's function. The purpose of this study was to conclusively determine the oligomerization state of these mutants using X-ray crystallography, NMR spectroscopy, thermal stability analysis, circular dichroism spectroscopy, sedimentation equilibrium ultracentrifugation, and cross-linking. We found that all of the Glu18 DJ-1 mutants were dimeric. Thiol cross-linking indicates that these mutant dimers are more flexible than the wild-type protein and can form multiple cross-linked dimeric species due to the transient exposure of cysteine residues that are inaccessible in the wild-type protein. The enhanced flexibility of Glu18 DJ-1 mutants provides a parsimonious explanation for their lower observed cross-linking efficiency in cells. In addition, thiol cross-linkers may have an underappreciated value as qualitative probes of protein conformational flexibility. DJ-1 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ-1 dimerization. We use cysteine cross-linking and various biophysical techniques to show that these DJ-1 mutants form dimers with increased conformational flexibility.
| S-EPMC4156530 | biostudies-literature
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