Biomedical subjects

Klavs B Hendil

Publications and source records attributed to Klavs B Hendil.

11 recordsLinked to original sources

PubMed2006

Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes.

The 20S proteasome is a catalytic core of the 26S proteasome, a central enzyme in the degradation of ubiquitin-conjugated proteins. It is composed of 14 distinct gene products that form four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). It is reported that the biogenesis of mammalian 20S proteasomes is assisted by proteasome-specific chaperones, named PAC1, PAC2, and hUmp1, but the details are still unknown. Here, we report the identification of a chaperone, designated PAC3, as a component of alpha rings. Although it can intrinsically bind directly to both alpha and beta subunits, PAC3 dissociates before the formation of half-proteasomes, a process coupled with the recruitment of beta subunits and hUmp1. Knockdown of PAC3 impaired alpha ring formation. Further, PAC1/2/3 triple knockdown resulted in the accumulation of disorganized half-proteasomes that are incompetent for dimerization. Our results describe a cooperative system of multiple chaperones involved in the correct assembly of mammalian 20S proteasomes.

Cell Line↗

PubMed2006

Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor.

We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function.

Amino Acid Sequence↗

PubMed2005

A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes.

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes. The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7 (refs 4, 5), but the mechanism responsible for the assembly of such a complex structure remains elusive. Here we report two chaperones, designated proteasome assembling chaperone-1 (PAC1) and PAC2, that are involved in the maturation of mammalian 20S proteasomes. PAC1 and PAC2 associate as heterodimers with proteasome precursors and are degraded after formation of the 20S proteasome is completed. Overexpression of PAC1 or PAC2 accelerates the formation of precursor proteasomes, whereas knockdown by short interfering RNA impairs it, resulting in poor maturation of 20S proteasomes. Furthermore, the PAC complex provides a scaffold for alpha-ring formation and keeps the alpha-rings competent for the subsequent formation of half-proteasomes. Thus, our results identify a mechanism for the correct assembly of 20S proteasomes.

Animals↗

PubMed2005

Development and use of antiproteasome monoclonal antibodies.

This chapter describes the production of hybridomas and screening of monoclonal antibodies for use in enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunoprecipitation of components of the ubiquitin-proteasome pathway. Purification of antibodies and their use in affinity chromatography are also described.

Animals↗

PubMed2004

The Ubx2 and Ubx3 cofactors direct Cdc48 activity to proteolytic and nonproteolytic ubiquitin-dependent processes.

Valosin-containing protein, VCP/p97 or Cdc48, is a eukaryotic ATPase involved in membrane fusion, protein transport, and protein degradation. We describe two proteins, Ubx2 and Ubx3, which interact with Cdc48 in fission yeast. Ubx3 is the ortholog of p47/Shp1, a previously described Cdc48 cofactor involved in membrane fusion, whereas Ubx2 is a novel protein. Cdc48 binds the UBX domains present in both Ubx2 and Ubx3, indicating that this domain is a general Cdc48-interacting module. Ubx2 and Ubx3 also interact with ubiquitin chains. Disruption of the ubx3(+)-gene causes both temperature and canavanine sensitivity and stabilizes some ubiquitin-protein conjugates including the CDK inhibitor Rum1, but not a model substrate of the ER-degradation pathway. Moreover the ubx3 null displays synthetic lethality with a pus1 null mutant, a multiubiquitin binding subunit of the 26S proteasome. In contrast, the ubx2 null mutant did not display any obvious protein-degradation phenotype. In conclusion Ubx3/p47 is not, as previously thought, only important for membrane fusion; it's also important for the specific degradation of a subset of cell proteins. Our genetic analyses revealed that Ubx3/p47 functionally parallels a substrate receptor of the 26S proteasome, Pus1/Rpn10, indicating that the Cdc48-Ubx3 complex is involved in delivering substrates to the 26S proteasome.

Adenosine Triphosphatases↗

PubMed2004

Proteasomal inhibition by alpha-synuclein filaments and oligomers.

A unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing alpha-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that alpha-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric alpha-synuclein. Proteasome binding to insoluble alpha-synuclein filaments and soluble alpha-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Abeta peptide aggregates. Endogenous ligands of aggregated alpha-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention.

Chymotrypsin↗

PubMed2004

Proteasomes: a complex story.

Protein degradation in eukaryotic cells is important for regulation of metabolism, progression through the division cycle, in cell signalling pathways, and in mammals also for generation of antigen fragments for presentation on the major histocompatibility complex (MHC) class I. Most cell proteins are degraded via the ubiquitin/proteasome pathway where an elaborate enzyme system recognises the protein substrates and marks them for destruction by attachment of a chain of ubiquitin. The substrates are then bound to 26S proteasomes, unfolded, and threaded into the cylindrical central part of the 26S proteasome, where they are cleaved to peptides. Recently many proteins, which associate with proteasomes, have been found. One of them controls the cellular contents of proteasomes by regulating their synthesis. Others ubiquitylate substrates or transfer substrates to proteasomes. Others again seem to unfold the substrates or release ubiquitin and glycans from them during degradation, stabilise proteasomes, regulate their cellular localisation, and modify their activity. It therefore appears that proteasomes are centres in macromolecular clusters, which degrade cell proteins in a tightly regulated manner.

Animals↗

PubMed2003

Ubiquitin binding proteins protect ubiquitin conjugates from disassembly.

As a step in their turnover proteins in eukaryotic cells are coupled to a small protein, ubiquitin, before they are recognised by 26S proteasomes and degraded. However, cells also contain many deubiquitinating enzymes, which can rescue proteins by cleaving off the ubiquitin chains. Here we report that three ubiquitin binding proteins, Rhp23, Dph1 and Pus1, from fission yeast can protect multiubiquitin conjugates against deubiquitination. This protection depends on the ubiquitin binding domains and may promote degradation of ubiquitinated proteins.

Amino Acid Motifs↗

PubMed2003

UBA domain containing proteins in fission yeast.

The ubiquitin-proteasome pathway for intracellular proteolysis is involved in a series of cellular and molecular functions, including the degradation of bulk proteins, cell cycle control, DNA repair, antigen presentation, vesicle transport and the regulation of signal transudation pathways and transcription. Considering this variety of cell biological processes, it is puzzling that until recently only very few proteins were known to possess the ability to interact specifically with ubiquitin chains. However, several ubiquitin binding proteins have now been identified and the binding domains have been characterised on both the functional and structural levels. One example of a widespread ubiquitin binding module is the ubiquitin associated (UBA) domain. Here, we discuss the approximately 15 UBA domain containing proteins encoded in the relatively small genome of the fission yeast Schizosaccharomyces pombe. The proteins display remarkable differences in their domain organisation, indicating that these potential ubiquitin binding proteins are involved in various cell activities.

Amino Acid Sequence↗

PubMed2002

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma.

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr approximately 30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8-3.3 and 3.0-3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose-response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r(2) of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319+/-237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.

Animals↗

PubMed2002

26 S proteasomes function as stable entities.

Most proteins in eukaryotic cells are degraded by 26-S proteasomes, usually after being conjugated to ubiquitin. In the absence of ATP, 26-S proteasomes fall apart into their two sub-complexes, 20-S proteasomes and PA700, which reassemble upon addition of ATP. Conceivably, 26-S proteasomes dissociate and reassemble during initiation of protein degradation in a ternary complex with the substrate, as in the dissociation-reassembly cycles found for ribosomes and the chaperonin GroEL/GroES. Here we followed disassembly and assembly of 26-S proteasomes in cell extracts as the exchange of PA700 subunits between mouse and human 26-S proteasomes. Compared to the rate of proteolysis in the same extract, the disassembly-reassembly cycle was much too slow to present an obligatory step in a degradation cycle. It has been suggested that subunit S5a (Mcb1, Rpn10), which binds poly-ubiquitin substrates, shuttles between a free state and the 26-S proteasome, bringing substrate to the complex. However, S5a was not found in the free state in HeLa cells. Besides, all subunits in PA700, including S5a, exchanged at similar low rates. It therefore seems that 26-S proteasomes function as stable entities during degradation of proteins.

Animals↗