Modulation of the Multicatalytic Proteinase Complex by Lipids, Interconversion and Proteolytic Processing

Arizti, Paz; Arribas, Joaquı́n; Castaño, José G.

doi:10.1159/000468686

Cited by 15 publications

(7 citation statements)

References 11 publications

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“…Except for this exchange no other consistent alterations in the two-dimensional pattern of the 20 S proteasome were noted. The significant acidic shift of the subunit C8 (30 kDa, basic of delta) in the double transfectant might be due to phosphorylation (45), but as it was not observed in a second preparation we did not further investigate this issue. The size and isoelectric point of the overexpressed LMP2 and LMP7 proteins in single and double transfectants are identical to those of the endogenously expressed proteins.…”

Section: Resultsmentioning

confidence: 99%

The Interferon-γ-inducible 11 S Regulator (PA28) and the LMP2/LMP7 Subunits Govern the Peptide Production by the 20 S Proteasome in Vitro

Groettrup

Ruppert

Kuehn

et al. 1995

Journal of Biological Chemistry

217

179

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Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-␥, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells.

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Section: Resultsmentioning

confidence: 99%

The Interferon-γ-inducible 11 S Regulator (PA28) and the LMP2/LMP7 Subunits Govern the Peptide Production by the 20 S Proteasome in Vitro

Groettrup

Ruppert

Kuehn

et al. 1995

Journal of Biological Chemistry

217

179

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“…Therefore, we suspect that an enrichment of the microsomal fraction with other membranes resulted in the high content of proteasomes. Another culprit may be activation of CP by membrane components (Arizti et al, 1993). its posttranslational modifications or a presence of CP distinctively decorated with activator modules (Hanna and Finley, 2007).…”

Section: Discussionmentioning

confidence: 99%

Molecular mechanisms of proteasome plasticity in aging

Rodriguez

Gaczyńska

Osmulski

2010

Mechanisms of Ageing and Development

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The ubiquitin-proteasome pathway plays a crucial role in regulation of intracellular protein turnover. Proteasome, the central protease of the pathway, encompasses multisubunit assemblies sharing a common catalytic core supplemented by regulatory modules and localizing to different subcellular compartments. To better comprehend age-related functions of the proteasome we surveyed content, composition and catalytic properties of the enzyme in cytosolic, microsomal and nuclear fractions. obtained from mouse livers subjected to organismal aging. We found that during aging subunit composition and subcellular distribution of proteasomes changed without substantial alterations in the total level of core complexes. We observed that the general decline in proteasomes functions was limited to nuclear and cytosolic compartments. Surprisingly, the observed changes in activity and specificity were linked to the amount of the activator module and distinct composition of the catalytic subunits. In contrast, activity, specificity and composition of the microsomal-associated proteasomes remained mostly unaffected by aging; however their relative contribution to the total activity was substantially elevated. Unexpectedly, the nuclear proteasomes were affected most profoundly by aging possibly triggering significant changes in cellular signaling and transcription. Collectively, the data indicate an age-related refocusing of proteasome from the compartment specific functions towards general protein maintenance.

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“…From the position of the phosphorylated proteasome subunits on two-dimensional PAGE gels, in the present study, it is clear that C8 might be the single 30-kDa subunit recently reported to be phosphorylated in vitro by a casein kinase I1 activity found in purified human erythrocyte proteasome preparations 1371. Casein kinase 11 activity in purified rat liver proteasome preparations has been reported to phosphorylate both C8 and C9 in v i m [53]. The amino acid sequence of C8 but not that of C9 contains a number of putative casein kinase I1 phosphorylation sites.…”

Section: Discussionmentioning

confidence: 99%

“…The protein levels of 26s proteinase/proteasome were approximately 1 : 2 as assessed by immunoblotting with antLC9 MCP257. in v i m [53]. The amino acid sequence of C8 but not that of C9 contains a number of putative casein kinase I1 phosphorylation sites.…”

Section: Substratementioning

confidence: 99%

Phosphorylation of Proteasomes in Mammalian Cells

Mason

Hendil²,

Rivett

1996

European Journal of Biochemistry

117

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The proteasome, a multimeric protease, plays an important role in nonlysosomal pathways of intracel-M a r protein degradation. This study was undertaken to determine which subunits of mammalian proteasomes are phosphorylated and to investigate the possible role of phosphorylation in regulating proteasome activity and the association with regulatory components. Rat-I fibroblasts were grown in the presence of ['*P]phosphate and proteasomes were immunoprecipitated from cell lysates with proteasome-specific polyclonal antibodies. Subsequent analysis by two-dimensional polyacrylamide gel electrophoresis showed two radiolabeled proteasome subunits which were identified using monoclonal antibodies as C8 and C9. Treatment of human embryonic lung cells (L-132), under identical conditions, also showed the same two phosphorylated subunits. Phosphoamino acid analysis revealed phosphoserine to be present in both C8 and C9. Examination of the sequence of C9 showed a potential cCMP-dependent phosphorylation site (-Arg3-Arg-Tyr-Asp-&-Arg8-), whilst C8 contains several potential casein kinase I1 phosphorylation sites. Following immunoprecipitation by a monoclonal antibody and dephosphorylation by acid phosphatase, proteasomes were observed to have significantly lower activities when compared to phosphorylated proteasomes, implying that phosphorylation may be an important mechanism of regulating proteasome function. Free proteasomes were separated by gel-filtration from those complexed with regulatory complexes to form the 26s proteinase. The ratio of phosphorylation of C8 and C9 was found to be very similar in the two complexes but the level of phosphorylation was higher in the 26s proteinase than in free proteasomes.Keywords: proteasome; 26s protease; phosphorylation ; pcptidase activity ; regulation.Proteasomes (multicatalytic proteinase complexes) are highmolecular-mass (700 kDa) multi-subunit cylindrical particles which are found in the nucleus and cytoplasm of eukaryotic cells (for reviews see [I -41). Eukaryotic proteasomes are composed of at least 14 non-identical subunits which are encoded by members of the same gene family. They can be divided into two groups a and p, based on their relationship to the a and /I subunits, respectively, of the simpler proteasome isolated from Abbrevintiorzs.AAF-NH-Mec, Ala-Ala-Phe-7-amino-4-1nethyl-coumarin; Boc-LSTR-NH-Mec, terf-butoxycarbonyl-Leu-Ser-Thr-Arg-7-amino-4-methylcoumarin; DMEM, Dulbecco's modified Eagle's medium; FCS, foetal calf serum; M-DMEM, methionine-free DMEM ; MHC, major histocornpatability complex: NaCIP,, 137 mM NaCI, 1.5 mM KH,PO,, 20 mM Na,HPO,, 2.7 mM KCI, pH 7.2; P-DMEM, phosphate-free DMEM: RIPA, 50 mM Tris pH 8.0, 150 mM NaCI, 1 % (masslvol.) Nonidet P-40, 0.1 % SDS, 0.5% sodium deoxycholate; Suc-LLVY-NH-Mec, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin: TrislNaCUEDTA. 50 mM Tris/HCl pH 7.0,150 mM NaCl, 5 mM EDTA, 0.1 % BSA, 0.1 % SDS, 0.1 % sodium deoxycholate, 0.5% (masslvol.) Nonidet-P40.Enzynrs. Proteasome (EC 3.4.99.46) ; potato acid phosphatase (EC ...

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Modulation of the Multicatalytic Proteinase Complex by Lipids, Interconversion and Proteolytic Processing

Cited by 15 publications

References 11 publications

The Interferon-γ-inducible 11 S Regulator (PA28) and the LMP2/LMP7 Subunits Govern the Peptide Production by the 20 S Proteasome in Vitro

The Interferon-γ-inducible 11 S Regulator (PA28) and the LMP2/LMP7 Subunits Govern the Peptide Production by the 20 S Proteasome in Vitro

Molecular mechanisms of proteasome plasticity in aging

Phosphorylation of Proteasomes in Mammalian Cells

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