The contribution of the main proteolytic pathways to the degradation of long-lived proteins in human fibroblasts grown under different conditions was investigated. The effects of various commonly used pharmacological inhibitors of protein degradation were first analysed in detail. By choosing specific inhibitors of lysosomes and proteasomes, it was observed that together both pathways accounted for 80% or more of the degradation of cell proteins. With lysosomal inhibitors, it was found that serum withdrawal or amino-acid deprivation strongly stimulated macroautophagy but not other lysosomal pathways, whereas confluent conditions had no effect on macroautophagy and slightly activated other lysosomal pathways. Prolonged (24 h) serum starvation of confluent cultures strongly decreased the macroautophagic pathway, whereas the activity of other lysosomal pathways increased. These changes correlated with electron microscopic observations and morphometric measurements of lysosomes. With proteasomal inhibitors, it was found that, in exponentially growing cells in the absence of serum, activity of the ubiquitin–proteasome pathway increases, whereas under confluent conditions the contribution (in percentage) of proteasomes to degradation decreases, especially in cells deprived of amino acids. Interestingly, in confluent cells, the levels of two components of the 19 S regulatory complex and those of an interchangeable β-subunit decreased. This was associated with a marked increase in the levels of components of PA28-immunoproteasomes. Thus confluent conditions affect proteasomes in a way that resembles treatment with interferon-γ. Altogether, these results show that the activity of the various proteolytic pathways depends on the growth conditions of cells and will be useful for investigation of the specific signals that control their activity.
Skip Nav Destination
Article navigation
October 2003
- PDF Icon PDF LinkFront Matter
Research Article|
October 01 2003
Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions
Graciela FUERTES;
Graciela FUERTES
∗Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya 4, 46010-Valencia, Spain
Search for other works by this author on:
José Javier MARTÍN DE LLANO;
José Javier MARTÍN DE LLANO
∗Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya 4, 46010-Valencia, Spain
Search for other works by this author on:
Adoración VILLARROYA;
Adoración VILLARROYA
∗Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya 4, 46010-Valencia, Spain
Search for other works by this author on:
A. Jennifer RIVETT;
A. Jennifer RIVETT
†Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS81TD, U.K.
Search for other works by this author on:
Erwin KNECHT
Erwin KNECHT
1
∗Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya 4, 46010-Valencia, Spain
1To whom correspondence should be addressed (e-mail knecht@ochoa.fib.es).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
February 19 2003
Revision Received:
June 23 2003
Accepted:
July 04 2003
Accepted Manuscript online:
July 04 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 375 (1): 75–86.
Article history
Received:
February 19 2003
Revision Received:
June 23 2003
Accepted:
July 04 2003
Accepted Manuscript online:
July 04 2003
Citation
Graciela FUERTES, José Javier MARTÍN DE LLANO, Adoración VILLARROYA, A. Jennifer RIVETT, Erwin KNECHT; Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions. Biochem J 1 October 2003; 375 (1): 75–86. doi: https://doi.org/10.1042/bj20030282
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.