Subunit arrangement in the human 20S proteasome

Köpp, F.; Hendil, Klavs B.; Dahlmann, Burkhardt; Kristensen, Poul; Sobek, Axel; Uerkvitz, Wolfgang

doi:10.1073/pnas.94.7.2939

Cited by 108 publications

(77 citation statements)

References 48 publications

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“…MG132 at this concentration also provoked the accumulation of prepro-PTH, whereas the effects of ALLM were the weakest, in agreement with its mild effects on proteasome activity. Furthermore, assay of the ATP-dependent chymotrypsin-like and peptidylglutamyl-peptide hydrolase proteasome activities in crude lysates from cells treated with the inhibitors used in this study confirmed the potency of MG132 (data not shown) and previous reports describing the differential potency of the cysteine proteinase inhibitors on the proteasome in other cells (42,43).…”

Section: Inhibition Of Parathyroid Proteasomes Leads To Accumulation supporting

confidence: 74%

Biosynthesis and Secretion of Parathyroid Hormone Are Sensitive to Proteasome Inhibitors in Dispersed Bovine Parathyroid Cells

Sakwe

Engström

Larsson

et al. 2002

Journal of Biological Chemistry

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Preproparathyroid hormone (prepro-PTH) is one of the proteins abundantly synthesized by parathyroid chief cells; yet under normal growth conditions, little or no prepro-PTH can be detected in these cells. Although this may be attributed to effective cotranslational translocation and proteolytic processing, proteasome-mediated degradation of PTH precursors may be important in the regulation of the levels of these precursors and hence PTH secretion. The effects of N-acetyl-Leu-Leunorleucinal, N-acetyl-Leu-Leu-methional, carbobenzoxy-Leu-Leu-leucinal (MG132), benzyloxycarbonyl-IleGlu(t-butyl)-Ala-leucinal (proteasome inhibitor I), and lactacystin on the biosynthesis and secretion of PTH were examined in dispersed bovine parathyroid cells. We demonstrate that treatment of these cells with proteasome inhibitors caused the accumulation of prepro-PTH and pro-PTH. Compared with mock-treated cells, the processing of pro-PTH to PTH was delayed, and the secretion of intact PTH decreased in proteasome inhibitor-treated cells. Relieving the inhibition of the proteasome by chasing MG132-treated cells in medium without the inhibitor led to the rapid disappearance of the accumulated prepro-PTH, and the rate of PTH secretion was restored to levels comparable to those in mocktreated cells. Furthermore, overexpression of the Hsp70 family of molecular chaperones was observed in proteasome inhibitor-treated cells, and we show that PTH/PTH precursors interact with these molecular chaperones. These data suggest the involvement of parathyroid cell proteasomes in the quality control of PTH biosynthesis.The calcium concentration in mammalian body fluids is tightly regulated predominantly by the actions of parathyroid hormone (PTH) 1 on bone, kidney, and intestine (1-4 ] o ) secretion of PTH. However, the signaling pathway(s) leading to the rapid and specific changes in PTH secretion remains largely unknown.The primary precursor prepro-PTH is translocated into the endoplasmic reticulum (ER) and cleaved to pro-PTH within 1 min. Pro-PTH then transits the ER and attains the trans-Golgi network within 20 min, where it is cleaved to mature PTH. Depending on the needs and predominantly on [Ca 2ϩ ] o , mature PTH is packaged into either secretory granules for exocytosis or storage granules. Secretion of de novo synthesized PTH is believed to occur within 30 min of prepro-PTH formation (6) and constitutes the bulk of secreted PTH (7-9). At the posttranscriptional level, acute changes in [Ca 2ϩ ] o (Ͻ24 h) do not significantly affect the rate of PTH biosynthesis. However, sustained or chronic hypocalcemia and hypercalcemia affect the stability and hence the levels of PTH mRNA (10 -14, 44).Intracellular proteolysis of mature bioactive PTH has been reported to be one of the mechanisms by which parathyroid cells regulate the amount of hormone available for secretion in response to changes in [Ca 2ϩ ] o (15,16). This PTH metabolism is now known to be mediated by calpains (17) and cathepsins B and D (18 -21). Inhibition of these PTH-degrading ac...

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Section: Inhibition Of Parathyroid Proteasomes Leads To Accumulation supporting

confidence: 74%

Biosynthesis and Secretion of Parathyroid Hormone Are Sensitive to Proteasome Inhibitors in Dispersed Bovine Parathyroid Cells

Sakwe

Engström

Larsson

et al. 2002

Journal of Biological Chemistry

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“…This antibody reacts with the phylogenetically preserved probox 1 motif shared by all -type subunits (HC2, HC3, HC8, HC9, Iota and Zeta); it reacts with proteasomes of several species, including higher plants, when tested on immunoblots of SDS/PAGE gels, yielding two main bands of 29 and 32 kDa (Hendil et al, 1995;Kopp et al, 1997;Tanaka & Tsurumi, 1997). A rabbit polyclonal antibody against the 20S proteasome "core" was obtained from Affiniti.…”

Section: Antibodiesmentioning

confidence: 99%

“…Western blotting shows bands attributable to "core" subunits at 25-30 kDa. This antibody has been shown to react with human, mouse and yeast proteasomes (Kopp et al, 1997;Tanaka & Tsurumi, 1997). Rabbit immunserum against liver rat proteasomes was a generous gift from B. Friguet (Conconi et al, 1996).…”

Section: Antibodiesmentioning

confidence: 99%

Evidence for the existence of a proteasome inToxoplasma gondii: intracellular localization and specific peptidase activities

et al. 2001

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Summary :The proteasome is a large intracellular protein complex whose main function is proteolytic removal of damaged proteins. It has recently been shown that the proteasome has a crucial role in the pathogenesis of protozoan parasites. We attempted to characterize the proteasome of T. gondii (RH strain). In immunoblot experiments, we showed that MCP231 monoclonal antibody, directed against the human 20S proteasome, labelled homologous proteins in T. gondii with a pattern similar to that observed in mammalian cells. The study of in vitro proteolytic activities showed that chymotrypsin-like activity (the only activity obtained with archaebacteria) was present in Toxoplasma, with K m and specific activity values close to those observed with eukaryotic cells. Immunofluorescence studies showed that the Toxoplasma proteasome predominated in the cytosol. KEY WORDS :Toxoplasma gondii, proteasome, immunoblotting, chymotrypsinlike, trypsin-like, fluorescent antibody. Résumé : MISE EN ÉVIDENCE DU PROTÉASOME DE TOXOPLASMA GONDII-. LOCALISATION INTRACELLULAIRE ET ACTIVITÉS SPÉCIFIQUES PEPTIDASIQUES

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“…In the fully assembled 20 S proteasome, each ␣-and ␤-subunit occupies a defined location relative to other subunits (7,17). Although the mechanism and orchestration of assembly is not well understood, it appears to occur in at least two stages, the first being the formation of a stable preproteasome complex (13-16 S) composed of an intact seven-member ␣-ring with at least three ␤ subunits (Z/␤2, C10-II/␤3, and C7-I/␤4) (18,19).…”

mentioning

confidence: 99%

Novel Propeptide Function in 20 S Proteasome Assembly Influences β Subunit Composition

Kingsbury

Griffin

Colbert

2000

Journal of Biological Chemistry

101

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The assembly of eukaryotic 20 S proteasomes involves the formation of half-proteasomes where precursor ␤-type subunits gather in position on an ␣-subunit ring, followed by the association of two half-proteasomes and ␤-subunit processing. In vertebrates three additional ␤-subunits (␤1i/LMP2, ␤2i/MECL1, and ␤5i/LMP7) can be synthesized and substituted for constitutive homologues (␤1/delta, ␤2/Z, and ␤5/X) to yield immunoproteasomes, which are important for generating certain antigenic peptides. We have shown previously that when all six ␤-subunits are present, cooperative assembly mechanisms limit the diversity of proteasome populations. Specifically, LMP7 is incorporated preferentially over X into preproteasomes containing LMP2 and MECL1. We show here that the LMP7 propeptide is responsible for this preferential incorporation, and it also enables LMP7 to incorporate into proteasomes containing delta and Z. In contrast, the X propeptide restricts incorporation to proteasomes with delta and Z. Furthermore, we demonstrate that the LMP7 propeptide can function in trans when expressed on LMP2, and that its NH 2 -terminal and mid-regions are particularly critical for function. In addition to identifying a novel propeptide function, our results raise the possibility that one consequence of LMP7 incorporation into both immunoproteasomes and delta/Z proteasomes may be to increase the diversity of antigenic peptides that can be generated.

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Subunit arrangement in the human 20S proteasome

Cited by 108 publications

References 48 publications

Biosynthesis and Secretion of Parathyroid Hormone Are Sensitive to Proteasome Inhibitors in Dispersed Bovine Parathyroid Cells

Biosynthesis and Secretion of Parathyroid Hormone Are Sensitive to Proteasome Inhibitors in Dispersed Bovine Parathyroid Cells

Evidence for the existence of a proteasome inToxoplasma gondii: intracellular localization and specific peptidase activities

Novel Propeptide Function in 20 S Proteasome Assembly Influences β Subunit Composition

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