<p>Proteolysis of short-lived cellular proteins was investigated in early- and late-passage cells from normal donors and in cells from donors with the premature aging diseases of progeria and the Werner syndrome. Studies were conducted using intact cells or crude cell extracts to measure the degradation of cellular proteins pre-labeled for 1 hour with ³H- and ¹⁴C-Iabeled phenylalanine.</p> <p>The purpose of these studies was:</p> <p>1. To determine whether aging cells (late-passage normal, progeria and Werner) show increased proteolysis compared to early-passage normals as would be predicted from the Orgel hypothesis.</p> <p>2. To investigate the effect of growth state and inhibitors on proteolysis in these cells.</p> <p>3. To identify specific cell fractions or proteins which undergo increased proteolysis in aging cells.</p> <p>4. To measure proteolysis of cellular proteins in extracts of cells, using both endogenous cellular proteases and added exogenous proteases. This was to probe the role of cellular proteins as substrates for prot eases in elevated proteolysis in aging cells.</p> <p>Proteolysis was increased in late-passage normal, progeria and Werner cells compared to early-passage normal cells. This was found in growth and at confluence. The chymotrypsin inhibitor TPCK reduced the elevated proteolysis in freshly plated late-passage and progeria cells but did not have this effect on cultures growing logarithmically or those at confluence. Increased proteolysis of soluble (post-microsomal) proteins may be occurring in progeria cells but SDS-polyacrylamide gels failed to show increased turnover of any protein bands.</p> <p>Protease activity in cell extracts could only be demonstrated at acid pH with endogenous cellular proteases. This proteolysis did not preferentially degrade proteins which have short half-lives in vivo including those containing the amino acid analogue canavanine. Proteins from early- and late-passage normal cells showed no difference in acid optimum proteolysis. However, proteins from progeria cells showed less proteolysis than normal controls. This was observed for labeled progeria proteins with proteases from extracts of normal or progeria cells. Paradoxically, proteins from progeria cells were more susceptible to labeled in the trypsin than normal controls but when proteins were labeled in the presence of TPCK no difference in trypsin susceptibility was observed.</p> <p>The finding indicate:</p> <p>1. Increased proteolysis is a feature of cellular aging.</p> <p>2. Proteolysis in aging cells during growth shows greater elevation compared to early-passage normals, than that seen at confluence.</p> <p>3. In progeria, cellular proteins have altered susceptibility to both endogenous and exogenous proteases.</p> <p>4. The inhibitor TPCK can, under certain conditions, reduce increased proteolysis in late-passage normal and progeria cells.</p> <p>Therefore, it is concluded that increased proteolysis occurs in aging cells and that increased activity of TPCK-sensitive proteases may in some circumstances be responsible. Thus, altered control of the proteolytic apparatus and not abnormal proteins may be causing increased proteolysis in aging cells.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/8013 |
| Date | 04 1900 |
| Creators | Elliot, John Joseph |
| Contributors | Goldstein, Samuel, Medical Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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