Assays of proteasome activity in relation to aging

Rivett, A. Jennifer; Bose, Suchira; Pemberton, Alexander J.; Brooks, Paul; Onion, David; Shirley, David G.; Stratford, Fiona L. L.; Forti, Katia

doi:10.1016/s0531-5565(02)00127-4

Cited by 31 publications

(29 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…We also examined the elution profile of UBE3C, another HECT Ub ligase, which has been previously shown to be associated with the proteasome (19,51,94). The fraction distribution of the proteasome in these experiments is similar to that described in the literature (7,28,70,74) and overlaps that of UBE3C (Fig. 2).…”

Section: Identification Of E6ap-interacting Proteinssupporting

confidence: 64%

Identification and Proteomic Analysis of Distinct UBE3A/E6AP Protein Complexes

Martínez-Noël

Galligan

Sowa

et al. 2012

Molecular and Cellular Biology

113

View full text Add to dashboard Cite

The E6AP ubiquitin ligase catalyzes the high-risk human papillomaviruses' E6-mediated ubiquitylation of p53, contributing to the neoplastic progression of cells infected by these viruses. Defects in the activity and the dosage of E6AP are linked to Angelman syndrome and to autism spectrum disorders, respectively, highlighting the need for precise control of the enzyme. With the exception of HERC2, which modulates the ubiquitin ligase activity of E6AP, little is known about the regulation or function of E6AP normally. Using a proteomic approach, we have identified and validated several new E6AP-interacting proteins, including HIF1AN, NEURL4, and mitogen-activated protein kinase 6 (MAPK6). E6AP exists as part of several different protein complexes, including the proteasome and an independent high-molecular-weight complex containing HERC2, NEURL4, and MAPK6. In examining the functional consequence of its interaction with the proteasome, we found that UBE3C (another proteasome-associated ubiquitin ligase), but not E6AP, contributes to proteasomal processivity in mammalian cells. We also found that E6 associates with the HERC2-containing high-molecular-weight complex through its binding to E6AP. These proteomic studies reveal a level of complexity for E6AP that has not been previously appreciated and identify a number of new cellular proteins through which E6AP may be regulated or functioning. E6AP was originally discovered as the cellular protein that mediates the binding of the E6 protein of the high-risk human papillomaviruses (HPVs) to the tumor suppressor p53 (35). Subsequently, E6AP was shown to catalyze the E6-dependent transfer of ubiquitin to p53, targeting it for degradation by the 26S proteasome. As such, E6AP was the first mammalian ubiquitin (Ub) ligase to be described (36,37,80).E6AP is a 100-kDa protein containing a conserved carboxyterminal domain spanning 350 amino acids, referred to as the HECT domain (homologous to E6AP carboxy terminus), that defines a family of E3 Ub ligases (34). This domain participates directly in the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a conserved cysteine residue through a thiolester bond (34). E6AP then directs the transfer of ubiquitin from this active-site cysteine to a lysine residue of an associated substrate. Replacement of the active-site cysteine with alanine renders E6AP unable to accept ubiquitin and therefore unable to catalyze the transfer of ubiquitin to the target protein. This C/A substitution mutant is inactive and functions in a dominant-negative (DN) manner (88). Three different isoforms of E6AP that are generated by differential splicing from the same gene have been described (98). These three isoforms differ in their amino-terminal sequences, and it is not yet known whether they differ in function or substrate specificity.The E6AP protein is encoded by the UBE3A gene located in an imprinted region on chromosome 15q11-q13. As a consequence of the imprinting, only the maternal UBE3A allele is expressed in certain areas of the br...

show abstract

Section: Identification Of E6ap-interacting Proteinssupporting

confidence: 64%

Identification and Proteomic Analysis of Distinct UBE3A/E6AP Protein Complexes

Martínez-Noël

Galligan

Sowa

et al. 2012

Molecular and Cellular Biology

113

View full text Add to dashboard Cite

show abstract

“…Extracts were centrifuged at 13,000 rpm for 10 min at 4°C, and then an equal amount of protein (1.5 mg) from empty vector or ␤ 5 subunit transfectants was fractionated by gel filtration using an Amersham Biosciences Superose 6 HR10/30 fast protein liquid chromatography column equilibrated in 20 mM Tris/HCl buffer, pH 7.5, containing 10% glycerol, 5 mM ATP, and 100 mM NaCl (36). Fractions were collected, and samples were analyzed by SDS-PAGE and immunoblot analysis.…”

Section: Preparation Of Cell Extracts and Separation Of Proteasome Comentioning

confidence: 99%

Overexpression of Proteasome β5 Assembled Subunit Increases the Amount of Proteasome and Confers Ameliorated Response to Oxidative Stress and Higher Survival Rates

Chondrogianni

Tzavelas

Pemberton

et al. 2005

Journal of Biological Chemistry

205

134

View full text Add to dashboard Cite

The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of ␤-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the ␤ 5 subunit. Stable overexpression of the ␤ 5 subunit in WI38/T and HL60 cells resulted in elevated levels of other ␤-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) ␣-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in ␤ 5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that ␤ 5 -overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the ␤ 5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4 -5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.

show abstract

“…Extracts were centrifuged at 11,500 ϫ g for 10 min at 4°C and then an equal amount of protein from early/late passage cells (0.5-1.5 mg) was fractionated by gel filtration using a Pharmacia Superose 6 FPLC column equilibrated in 20 mM Tris/HCl buffer, pH 7.5, containing 10% glycerol, 5 mM ATP, and 100 mM NaCl (34). Fractions were collected and samples were analyzed by SDS-PAGE and immunoblot analysis.…”

Section: Preparation Of Cell Extracts and Separation Of Proteasome Comentioning

confidence: 99%

Central Role of the Proteasome in Senescence and Survival of Human Fibroblasts

Chondrogianni

Stratford

Trougakos

et al. 2003

Journal of Biological Chemistry

Self Cite

299

111

View full text Add to dashboard Cite

Normal human fibroblasts undergo a limited number of divisions in culture and progressively they reach a state of irreversible growth arrest, a process termed as replicative senescence. The proteasome is the major cellular proteolytic machinery, the function of which is impaired during replicative senescence. However, the exact causes of its malfunction in these conditions are unknown. Using WI38 fibroblasts as a model for cellular senescence we have observed reduced levels of proteasomal peptidase activities coupled with increased levels of both oxidized and ubiquitinated proteins in senescent cells. We have found the catalytic subunits of the 20 S complex and subunits of the 19 S regulatory complex to be down-regulated in senescent cells. This is accompanied by a decrease in the level of both 20 S and 26 S complexes. Partial inhibition of proteasomes in young cells caused by treatment with specific inhibitors induced a senescence-like phenotype, thus demonstrating the fundamental importance of the proteasome for retaining cellular maintenance and homeostasis. Stable overexpression of ␤ 1 and ␤ 5 subunits in WI38 established cell lines was shown to induce elevated expression levels of ␤ 1 subunit in ␤ 5 transfectants and vice versa.

show abstract

Assays of proteasome activity in relation to aging

Cited by 31 publications

References 19 publications

Identification and Proteomic Analysis of Distinct UBE3A/E6AP Protein Complexes

Identification and Proteomic Analysis of Distinct UBE3A/E6AP Protein Complexes

Overexpression of Proteasome β5 Assembled Subunit Increases the Amount of Proteasome and Confers Ameliorated Response to Oxidative Stress and Higher Survival Rates

Central Role of the Proteasome in Senescence and Survival of Human Fibroblasts

Contact Info

Product

Resources

About