Overexpression of the two-chain form of cathepsin B in senescent WI-38 cells.
We have examined differential protein expression in serum-stimulated young and senescent WI-38 human fetal lung-derived cells in culture using high-resolution two-dimensional gel electrophoresis. Overexpression of a protein with an approximate M(r) of 29,000 and pI of 5.8 was observed in senescent cells during the G0 and throughout the G1 stage of the cell cycle. Automated amino-terminal sequencing of the peptide from polyvinylidene difluoride electroblots showed 100% sequence identity to cathepsin B or pre-procathepsin B in a 12-amino acid overlap, beginning at residue 48 or 129, respectively. The 29-kDa peptide corresponds to the heavy chain of the two-chain enzyme form. Cathepsin B activity was found to be decreased in cells aged in vitro in comparison to that in young controls. Changes in the steady-state levels of both the 4.0- and the 2.2-kb cathepsin B transcripts between young and senescent cells cannot account for the overexpression of the two-chain form of the enzyme. These results suggest that increased proteolysis of a conformationally more labile single-chain form and/or decreased turnover and accumulation of a less active form of this lysosomal protease occur in senescent fibroblasts and may account for the observed decreased cathepsin B activity in senescent cells in culture.
DiPaolo
B R
BR
Center for Gerontological Research, Medical College of Pennsylvania, Philadelphia 19129.
Pignolo
R J
RJ
Cristofalo
V J
VJ
D000595:Amino Acid Sequence; D002401:Cathepsin B / Q000032:analysis / Q000378:metabolism*; D002403:Cathepsins / Q000032:analysis; D002460:Cell Line; D016922:Cellular Senescence; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000032:analysis; D016193:G1 Phase; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D016192:Resting Phase, Cell Cycle
D004792:Enzyme Precursors; D012333:RNA, Messenger; D002403:Cathepsins; C057831:preprocathepsin B; D002401:Cathepsin B