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)

The conformational changes of human apolipoprotein (apo) B-100 which accompany the conversion of plasma very low density lipoproteins (VLDL) to low density lipoproteins (LDL) were investigated by studying the accessibility of apoB-100 in LDL and VLDL to limited proteolysis with cathepsin D, an aspartyl proteinase involved in intracellular protein degradation. We characterized the proteolytic products of apoB-100 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by NH2-terminal sequence analysis to locate cleavage sites. The results identified at least 10 cleavage products generated from apoB-100 and showed differential accessibility of cleavage sites for cathepsin D in apoB-100 between LDL and VLDL. We identified a specific peptide region (residues 2660-2710), which is preferentially accessible to limited proteolysis by cathepsin D but inaccessible to limited proteolysis by 12 other enzymes tested. Within this peptide region, cathepsin D cleaved apoB-100 of LDL and VLDL preferentially at different sites, separated by 33-36 amino acids (2665-2666 or 2668-2669 (LDL) and 2701-2702 (VLDL]. In addition, we identified a cleavage site, located at residues 3272-3273, specific for cathepsin D, which is contained within the COOH-terminal enzyme-accessible peptide region (residues 3180-3280), which we have demonstrated using 12 endoproteases with various specificities. The previously identified NH2-terminal region (residues 1280-1320) appears to be resistant to limited cleavage by cathepsin D. However, a new site was revealed only approximately 66 kDA from the NH2 terminus. We conclude that differential accessibility and the shift of the novel scission site for cathepsin D by 33-36 amino acids indicate significant differences in local conformation at these sites in apoB-100 as VLDL are converted to LDL.
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PMID:Differences in local conformation in human apolipoprotein B-100 of plasma low density and very low density lipoproteins as identified by cathepsin D. 206 28

The flanking sequences of several genes have been shown to direct a position independent expression of transgenes. Attempts to completely identify the insulating sequences have failed so far. Some of these sequences contain a matrix attached region (MAR) located in the flanking part of the genes. This article will show that the MARs in cultured cells located in the 3' OH region of the human apolipoprotein B100 (Apo B100) and within the SV40 genome were unable to stimulate and insultate transgene expression directed by the promoters from a rabbit whey acidic protein (WAP) gene or from human cytomegalovirus (hCMV) early genes. In transgenic mice, the MAR from the Apo B100 and SV40 genes did not enhance the expression of a transgene containing the rabbit whey acid protein (WAP) promotor, the late gene SV40 intron (VP1 intron), the bovine growth hormone (bGH) cDNA and the SV40 late gene terminator. This construct was even toxic for embryos. Similarly, the specialized chromatin structure (SCS) from the Drosophila 87A7 HSP70 gene reduced chloramphenicol acetyl transferase (CAT) activity when added between a cytomegalovirus (CMV) enhancer and a Herpes simplex thymidine kinase (TK) gene promoter. This inhibitory action was almost complete when a second SCS sequence was added before the CMV enhancer. Sequences from the firefly luciferase and from the human gene cathepsin D cDNA used as control unexpectedly showed a similar inhibitory effect when added to the CMVTKCAT construct instead of SCS. When added before the CMV enhancer and after the transcription terminator in the CMVTKCAT construct, the SCS sequence was unable to insulate the integrated gene as seen by the fact that the level of CAT in cell extracts were by no means correlated with the number of copies in individual clones. From these data, it is concluded that i) a MAR containing the canonical AT rich sequences does not amplify the expression of all gene constructs ii) At rich MAR sequences do not have per se an insulating effect iii) Drosophila SCS from the 87A7 HSP70 gene has no insulating effect in all gene constructs (at least in mammalian cells) iv) and the addition of a DNA fragment between an enhancer and a promoter in a gene construct cannot be used as a reliable test to evaluate its insulating property.
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PMID:The effect of matrix attached regions (MAR) and specialized chromatin structure (SCS) on the expression of gene constructs in cultured cells and in transgenic mice. 885 71