Phosphorylation of ATPase subunits of the 26S proteasome

Abstract The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2DPAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S oteasomes were immunoprecipitated as above and dissociated and Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable c-interferoninducible catalytic b-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit speci®c antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core a-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some signi®cant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immuno¯uorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following cinterferon treatment of cultured cells but c-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Proteasome inhibitors as anti-cancer agents

The ubiquitin (Ub)-proteasome pathway is the major nonlysosomal pathway of proteolysis in human cells and accounts for the degradation of most short-lived, misfolded or damaged proteins. This pathway is important in the regulation of a number of key biological regulatory mechanisms. Proteins are usually targeted for proteasome-mediated degradation by polyubiquitinylation, the covalent addition of multiple units of the 76 amino acid protein Ub, which are ligated to 1-amino groups of lysine residues in the substrate. Polyubiquitinylated proteins are degraded by the 26S proteasome, a large, ATP-dependent multicatalytic protease complex, which also regenerates monomeric Ub. The targets of this pathway include key regulators of cell proliferation and cell death. An alternative form of the proteasome, termed the immunoproteasome, also has important functions in the generation of peptides for presentation by MHC class I molecules. In recent years there has been a great deal of interest in the possibility that proteasome inhibitors, through elevation of the levels of proteasome targets, might prove useful as a novel class of anti-cancer drugs. Here we review the progress made to date in this area and highlight the potential advantages and weaknesses of this approach.

Proteasome activities decrease during dexamethasone-induced apoptosis of thymocytes

The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially puri®ed from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-¯uoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-¯uoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in .itro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.

Regulation of proteasome structure and function

Proteasomes are cylindrical particles made up of a stack of four heptameric rings. In animal cells the outer rings are made up of 7 different types of alpha subunits and the inner rings are composed of 7 out of 10 possible different beta subunits. Regulatory complexes can bind to the ends of the cylinder.We have investigated aspects of the assembly, activity and subunit composition of core proteasome particles and 26S proteasomes, the localization of proteasome subpopulations, and the possible role of phosphorylation in determining proteasome localization, activities and association with regulatory components.

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the c~ and /3 subunits of the simpler proteasome isolated from Thermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that /3-type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these/3-type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulated in vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.

Trauma and survival in African humanitarian entrants to Australia

Former refugees have been resettled in Australia since the 1940’s through the Humanitarian Migration Stream. This chapter highlights the impact of forced migration and the refugee experience of trauma on survival. The journey from pre-migration crises, to the process of fleeing one’s country, through to the challenges associated with resettlement, can have a significant impact on the mental health of Humanitarian Entrants to Australia. Differences in culture can have an impact on the meaning constructed from these experiences, and on help-seeking behaviour and preferred methods of intervention. To date, Western mental health services have used an understanding of trauma based on pathology and largely individualist intervention techniques. In this chapter, however, we seek to understand the experience of trauma for former refugees from a salutogenic perspective, and acknowledge community based coping methods and the strengths and resilience of former refugees. Using the construct of posttraumatic growth, adaptive factors of strength, religion, compassion, and new possibilities are identified as relevant to African Humanitarian Entrants in Australia.

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

Adverse childhood experiences and the health of university students in eight provinces of Vietnam

Recent systematic reviews have emphasized the need for more research into the health and social impacts of adverse childhood experiences (ACEs) in the Asia-Pacific region. This cross-sectional study was conducted with 2099 young adult students in 8 medical universities throughout Vietnam. An anonymous, self-report questionnaire included the World Health Organization ACE-International Questionnaire and standardized measures of mental and physical health. Three quarters (76%) of the students reported at least one exposure to ACEs; 21% had 4 or more ACEs. The most commonly reported adversities were emotional abuse, physical abuse, and witnessing a household member being treated violently (42.3%, 39.9%, and 34.6%, respectively). Co-occurrence of ACEs had dose–response relationships with poor mental health, suicidal ideation, and low physical health–related quality of life. This first multisite study of ACEs among Vietnamese university students provided evidence that childhood adversity is common and is significantly linked with impaired health and well-being into the early adult years

Evaluation of growth and changes in body composition following neonatal diagnosis of cystic fibrosis

Early deficits in nutritional status that might require specific treatment and early response to nutritional therapy were studied longitudinally in 25 infants with cystic fibrosis (CF) diagnosed by neonatal screening, using anthropometric and research body composition methodology, and evaluation of pancreatic function. At the time of confirmed diagnosis (mean 5.4 weeks), body mass, length, total body fat (TBF), and total body potassium (TBK) were all significantly reduced. Following diagnosis and commencement of therapy there was a normalization of weight, length, and TBK by 6-12 months of age, indicating catch-up growth. But in some individuals the response was incomplete, and as a group, mean total body fat remained significantly lower than normal at 1 year of age. Seven of 25 (28%) were pancreatic sufficient at diagnosis, and all but one had evidence of declining pancreatic function requiring the institution of pancreatic enzyme therapy during the next 1-9 months. The median age of commencement of enzyme therapy was 10 weeks (range 5 weeks to 11 months). These longitudinal assessments emphasize the dynamic changes occurring in absorptive function, body composition, and nutritional status following neonatal diagnosis of cystic fibrosis and may reflect previously described abnormalities of energy metabolism in this age group. Abnormal body composition is evident in most CF infants following diagnosis by neonatal screening but pancreatic damage may still be evolving. We suggest that early active nutritional therapy and surveillance for changes in pancreatic function are warranted in CF infants diagnosed by neonatal screening.