Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin.
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ABSTRACT:
SUBMITTER: Crawford C
PROVIDER: S-EPMC1148582 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
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Project description:1. Normal human serum was found to inhibit human cathepsin B1. 2. The major inhibitor present in serum was purified and identified as alpha(2)-macroglobulin. 3. alpha(2)-Macroglobulin was found to bind cathepsin B1 in an approximately 1:1 molar ratio. When bound, the enzyme retained about 50% of its proteolytic activity, and up to 80% of its activity against alpha-N-benzoyl-dl-arginine 2-naphthylamide. 4. Pretreatment of alpha(2)-macroglobulin with cathepsin B1 inactivated by exposure to pH8.5 or iodoacetic acid, in large molar excess, did not prevent the subsequent binding of active enzyme. Active enzyme, once bound, was not protected from inhibition by 1-chloro-4-phenyl-3-tosylamido-l-butan-2-one. 5. Cathepsin B1 was also inhibited by human immunoglobulin G, at high concentration. 6. Because it had been suggested that haptoglobin is responsible for the inhibition of ;cathepsin B' by serum, a method was devised for the selective removal of haptoglobin from mixtures of serum proteins by adsorption on haemoglobin covalently linked to Sepharose. No evidence was obtained that haptoglobin has any inhibitory activity against the enzyme.
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Project description:Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of alpha II-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The alpha II-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the alpha I-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in alpha II-spectrin. Western blotting showed that alpha II-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of alpha II-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.
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Project description:The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins (G, QxVxG) was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain (G, QxVxG, PW), a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn α-helix, a five stranded β-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.
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Project description:When chicken calpain II autolysed in the presence of Ca2+, it underwent limited proteolysis to give peptides of Mr 54,000 and 37,000, and several of Mr approx. 30,000 and 18,000. The autolytic peptides were purified and their N-terminal amino acid sequences determined. By comparison of these sequences with the known sequence of the complete calpain molecule, the autolytic cleavage sites were identified. The structural integrity of the molecule during autolysis was investigated by gel-permeation chromatography. Experiments were also done to test the reversibility of adding EDTA to calpain during autolysis, measured as recoverable enzyme activity assayed in the presence of Ca2+. The results are presented in terms of a model for the structural changes occurring in calpain during autolysis. It was concluded that the loss of enzymic activity, which is a consequence of autolysis, was due to dissociation of the autolytic peptides after cleavage of the calpain large subunit within the third domain.
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Project description:The effect of alpha 2-macroglobulin, one of the major antiproteinases in the plasma of vertebrates, on the action of the aspartic proteinases chymosin, cathepsin D and cathepsin E towards peptide and protein substrates at pH 6.2 was examined. Activities towards protein substrates were blocked, thus demonstrating that alpha 2-macroglobulin can inhibit aspartic proteinases, in addition to serine proteinases, cysteine proteinases and metalloproteinases.
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Project description:Extracellular protein misfolding is implicated in many age-related diseases including Alzheimer's disease, macular degeneration and arthritis. In this study, putative endogenous clients for the chaperone activity of α₂-macroglobulin (α₂M) were identified after human plasma was subjected to physiologically relevant sheer stress at 37 °C for 10 days. Western blot analysis showed that four major acute phase proteins: ceruloplasmin, fibrinogen, α₁-acid glycoprotein and complement component 3, preferentially co-purified with α₂M after plasma was stressed. Furthermore, the formation of complexes between α₂M and these putative chaperone clients, detected by sandwich ELISA, was shown to be enhanced in response to stress. These results support the hypothesis that α₂M plays an important role in extracellular proteostasis by sequestering misfolded proteins and targeting them for disposal, particularly during acute phase reactions.
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Project description:Proteostasis regulates protein folding and degradation; its maintenance is essential for resistance to stress and aging. The loss of proteostasis is associated with many age-related diseases. Within the cell, molecular chaperones facilitate the refolding of misfolded proteins into their bioactive forms, thus preventing undesirable interactions and aggregation. Although the mechanisms of intracellular protein degradation pathways for intracellular misfolded proteins have been extensively studied, the protein degradation pathway for extracellular proteins remain poorly understood. In this study, we identified several misfolded proteins that are substrates for alpha 2-macroglobulin (α2M), an extracellular chaperone. We also established a lysosomal internalization assay for α2M, which revealed that α2M mediates the lysosomal degradation of extracellular misfolded proteins. Comparative analyses of α2M and clusterin, another extracellular chaperone, indicated that α2M preferentially targets aggregation-prone proteins. Thus, we present the degradation pathway of α2M, which interacts with aggregation-prone proteins for lysosomal degradation via selective internalization.
| S-EPMC10050189 | biostudies-literature
Project description:Alpha-2-macroglobulin is an extracellular macromolecule mainly known for its role as a broad-spectrum protease inhibitor. By presenting itself as an optimal substrate for endopeptidases of all catalytic types, alpha-2-macroglobulin lures active proteases into its molecular cage and subsequently 'flags' their complex for elimination. In addition to its role as a regulator of extracellular proteolysis, alpha-2-macroglobulin also has other functions such as switching proteolysis towards small substrates, facilitating cell migration and the binding of cytokines, growth factors and damaged extracellular proteins. These functions appear particularly important in the context of immune-cell function. In this review manuscript, we provide an overview of all functions of alpha-2-macroglobulin and place these in the context of inflammation, immunity and infections.
| S-EPMC8712716 | biostudies-literature
Project description:Alpha-2-Macroglobulin (A2M) is the critical pan-protease inhibitor of the innate immune system. When proteases cleave the A2M bait region, global structural transformation of the A2M tetramer is triggered to entrap the protease. The structural basis behind the cleavage-induced transformation and the protease entrapment remains unclear. Here, we report cryo-EM structures of native- and intermediate-forms of the Xenopus laevis egg A2M homolog (A2Moo or ovomacroglobulin) tetramer at 3.7-4.1 Å and 6.4 Å resolution, respectively. In the native A2Moo tetramer, two pairs of dimers arrange into a cross-like configuration with four 60 Å-wide bait-exposing grooves. Each bait in the native form threads into an aperture formed by three macroglobulin domains (MG2, MG3, MG6). The bait is released from the narrowed aperture in the induced protomer of the intermediate form. We propose that the intact bait region works as a "latch-lock" to block futile A2M transformation until its protease-mediated cleavage.
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Project description:BackgroundThe cystatin superfamily comprises cysteine protease inhibitors that play key regulatory roles in protein degradation processes. Although they have been the subject of many studies, little is known about their genesis, evolution and functional diversification. Our aim has been to obtain a comprehensive insight into their origin, distribution, diversity, evolution and classification in Eukaryota, Bacteria and Archaea.ResultsWe have identified in silico the full complement of the cystatin superfamily in more than 2100 prokaryotic and eukaryotic genomes. The analysis of numerous eukaryotic genomes has provided strong evidence for the emergence of this superfamily in the ancestor of eukaryotes. The progenitor of this superfamily was most probably intracellular and lacked a signal peptide and disulfide bridges, much like the extant Giardia cystatin. A primordial gene duplication produced two ancestral eukaryotic lineages, cystatins and stefins. While stefins remain encoded by a single or a small number of genes throughout the eukaryotes, the cystatins have undergone a more complex and dynamic evolution through numerous gene and domain duplications. In the cystatin superfamily we discovered twenty vertebrate-specific and three angiosperm-specific orthologous families, indicating that functional diversification has occurred only in multicellular eukaryotes. In vertebrate orthologous families, the prevailing trends were loss of the ancestral inhibitory activity and acquisition of novel functions in innate immunity. Bacterial cystatins and stefins may be emergency inhibitors that enable survival of bacteria in the host, defending them from the host's proteolytic activity.ConclusionThis study challenges the current view on the classification, origin and evolution of the cystatin superfamily and provides valuable insights into their functional diversification. The findings of this comprehensive study provide guides for future structural and evolutionary studies of the cystatin superfamily as well as of other protease inhibitors and proteases.
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