Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.23.5 (cathepsin D)
 4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA encoding a Schistosoma japonicum aspartic proteinase was cloned, sequenced, and found to encode a zymogen of 380 amino acid residues, and its gene was shown to be present as a single copy in the S. japonicum genome. Identity comparisons showed that the enzyme (Sjpasp) was most closely related to the cathepsin Ds. The deduced amino acid sequence has four potential glycosylation sites, two of which are in identical positions to the two glycosylation sites of human kidney lysosomal cathepsin D. Furthermore, all four disulfide bonds found in mammalian cathepsin D sequences are present in Sjpasp, although the beta-hairpin (loop 3), which is cleaved during maturation of vertebrate cathepsin Ds to yield light and heavy chain subunits, is absent from Sjpasp. While most residues involved in substrate specificity and catalysis of aspartic proteinases are preserved in Sjpasp, several residues in these regions exhibit changes that may result in a novel substrate specificity. Aspartic proteinase activity is present in extracts of adult S. japonicum and Schistosoma mansoni and in culture media in which schistosomes were maintained and was capable of digesting hemoglobin. The schistosome aspartic proteinase may play a pivotal role in the catabolism of hemoglobin obtained from host erythrocytes.
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PMID:Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation. 759 66

Invasion of the cultured epithelial cell lines HeLa, Henle-407, and Caco-2 (polarized and nonpolarized) by Salmonella typhi and Salmonella typhimurium were compared using conventional gentamicin invasion assays. Additionally, the mechanisms of invasion and intracellular trafficking by S. typhi and S. typhimurium were compared in HeLa cells using indirect immunofluorescence microscopy. S. typhi strain Ty2 was invasive in all human cell lines tested, including apical uptake into polarized Caco-2 cell monolayers. This strain also replicated at levels similar to S. typhimurium strain SL1344 inside HeLa and Henle-407 cells. Indirect immunofluorescence microscopy confirmed that S. typhi, like S. typhimurium, induced membrane ruffles and cytoskeletal rearrangements upon contact with HeLa cell surfaces. Ruffling induced by S. typhi and S. typhimurium was accompanied by macropinocytosis of the fluid phase endocytic marker fluorescein-dextran-sulphate and by aggregation of cell surface class I MHC heavy chain. Intracellular lysosomal trafficking of S. typhi and S. typhimurium in HeLa cells was also studied. The lysosomal membrane glycoprotein marker h-lamp-2 colocalized with S. typhi-containing vacuoles, as previously shown for S. typhimurium. The soluble lysosomal enzyme marker cathepsin D also was found within S. typhi-containing vacuoles to the same extent as previously published for S. typhimurium. The results from this study suggest that S. typhi and S. typhimurium use similar mechanisms for invasion and intracellular trafficking in cultured human epithelial cells.
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PMID:Comparison of Salmonella typhi and Salmonella typhimurium invasion, intracellular growth and localization in cultured human epithelial cells. 775 82

Plasma cells secrete IgM only in the polymeric form: the C-terminal cysteine of the mu heavy chain (Cys575) is responsible for both intracellular retention and assembly of IgM subunits. Polymerization is not quantitative, and part of IgM is degraded intracellularly. Neither chloroquine nor brefeldin A (BFA) inhibits degradation, suggesting that this process occurs in a pre-Golgi compartment. Degradation of IgM assembly intermediates requires Cys575: the monomeric IgMala575 mutant is stable also when endoplasmic reticulum (ER) to Golgi transport is blocked by BFA. Addition of the 20 C-terminal residues of mu to the lysosomal protease cathepsin D is sufficient to induce pre-Golgi retention and degradation of the chimeric protein: the small amounts of molecules which exit from the ER are mostly covalent dimers. By contrast, when retained by the KDEL sequence, cathepsin D is stable in the ER, indicating that retention is not sufficient to cause degradation. Replacing the C-terminal cysteine with serine restores transport through the Golgi. As all chimeric cathepsin D constructs display comparable protease activity in vitro, their different fates are not determined by gross alterations in folding. Thus, also out of its normal context, the mu chain Cys575 plays a crucial role in quality control, mediating assembly, retention and degradation.
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PMID:Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation. 822 84

The mature form of cathepsin D (Cat D), purified to homogeneity from postmortem human brain or mouse brain, behaved as a 42-kDa protein in its native state but revealed additional proteolytic processing under denaturing conditions. Human brain Cat D was composed of a 30-32 kDa heavy chain and a protein doublet consisting of 14 and 15 kDa light chains. Mouse Cat D, which closely resembled the human enzyme in amino acid composition, existed mainly as the uncleaved 42-kDa protein, but up to 40% existed as a complex of 30-32 kDa and 12-14 kDa chains. The 3:1 ratio of light to heavy (30-32 kDa) chains suggested processing of some 30-kDa chains. Cleavage of the 42-kDa chain could not be induced autolytically. Human brain Cat D had a 2-3-fold higher specific activity than the mouse enzyme but shared other properties, including similar biphasic pH optima (peaks at pH 3.30 and 4.2), Km values for methemoglobin and inhibitor profiles. Human Cat D displayed the same polypeptide chain composition when purified from brains differing in postmortem interval (3-28 h). Fresh SH-SY5Y human neuroblastoma cells analyzed on Western blots with anti-Cat D antibodies also displayed only cleaved forms of mature Cat D. Furthermore, brain Cat D isolated from mice stored after death for 5, 15 or 30 h at 25 degrees C contained the same molar ratios of cleaved and uncleaved enzyme found in fresh mouse brain . Cat D activity was stable in human brains with postmortem intervals up to 27 h and stored frozen for up to 3 years. Similarly, total Cat D activity was essentially unchanged in brains of mice subjected to stimulated postmortem conditions for 0.5-4.2 h, although 20% of the total soluble brain protein became insoluble during this postmortem interval. These results demonstrate a remarkable postmortem stability of Cat D and strongly suggest that limited proteolytic cleavage of mature brain Cat D is an in vivo event, the extent of which varies markedly in different species.
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PMID:Limited proteolytic processing of the mature form of cathepsin D in human and mouse brain: postmortem stability of enzyme structure and activity. 884 39

Chinese hamster ovary (CHO) cells are often used to produce therapeutic monoclonal antibodies (mAbs). CHO cells express many host cell proteins (HCPs) required for their growth. Interactions of HCPs with mAbs can sometimes result in co-purification of trace levels of 'hitchhiker' HCPs during the manufacturing process. Purified mAb-1 product produced in early stages of process optimization had high HCP levels. In addition, these lots formed delayed-onset particles containing mAb-1 and its heavy chain C-terminal fragments. Studies were performed to determine the cause of the observed particle formation and to optimize the purification for improved HCP clearance. Protease activity and inhibitor stability studies confirmed that an aspartyl protease was responsible for fragmentation of mAb-1 resulting in particle formation. An affinity resin was used to selectively capture aspartyl proteases from the mAb-1 product. Mass spectrometry identified the captured aspartyl protease as CHO cathepsin D. A wash step at high pH with salt and caprylate was implemented during the protein A affinity step to disrupt the HCP-mAb interactions and improve HCP clearance. The product at the end of purification using the optimized process had very low HCP levels, did not contain detectable protease activity, and did not form particles. Spiking of CHO cathepsin D back into mAb-1 product from the optimized process confirmed that it was the cause of the particle formation. This work demonstrated that process optimization focused on removal of HCPs was successful in eliminating particle formation in the final mAb-1 product.
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PMID:Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product. 2625 61


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