Isolation of Mammalian 26S Proteasomes and p97/VCP Complexes Using the Ubiquitin-like Domain from HHR23B Reveals Novel Proteasome-Associated Proteins

Besche, Henrike C.; Haas, Wilhelm; Gygi, Steven P.; Goldberg, Alfred L.

doi:10.1021/bi802198q

Cited by 162 publications

(188 citation statements)

References 70 publications

(124 reference statements)

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“…The affinity purification of proteasomes using the UBL domain as an affinity ligand was performed in the absence of NaCl as described previously (7).…”

Section: Methodsmentioning

confidence: 99%

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Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

Altun

Besche

Overkleeft

et al. 2010

Journal of Biological Chemistry

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189

149

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Among the hallmarks of aged organisms are an accumulation of misfolded proteins and a reduction in skeletal muscle mass ("sarcopenia"). We have examined the effects of aging and dietary restriction (which retards many age-related changes) on components of the ubiquitin proteasome system (UPS) in muscle. The hindlimb muscles of aged (30 months old) rats showed a marked loss of muscle mass and contained 2-3-fold higher levels of 26S proteasomes than those of adult (4 months old) controls. 26S proteasomes purified from muscles of aged and adult rats showed a similar capacity to degrade peptides, proteins, and an ubiquitylated substrate, but differed in levels of proteasome-associated proteins (e.g. the ubiquitin ligase E6AP and deubiquitylating enzyme USP14). Also, the activities of many other deubiquitylating enzymes were greatly enhanced in the aged muscles. Nevertheless, their content of polyubiquitylated proteins was higher than in adult animals. The aged muscles contained higher levels of the ubiquitin ligase CHIP, involved in eliminating misfolded proteins, and MuRF1, which ubiquitylates myofibrillar proteins. These muscles differed from ones rapidly atrophying due to disease, fasting, or disuse in that Atrogin-1/MAFbx expression was low and not inducible by glucocorticoids. Thus, the muscles of aged rats showed many adaptations indicating enhanced proteolysis by the UPS, which may enhance their capacity to eliminate misfolded proteins and seems to contribute to the sarcopenia. Accordingly, dietary restriction decreased or prevented the aging-associated increases in proteasomes and other UPS components and reduced muscle wasting.

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“…The affinity purification of proteasomes using the UBL domain as an affinity ligand was performed in the absence of NaCl as described previously (7).…”

Section: Methodsmentioning

confidence: 99%

“…To assess their ability to degrade ubiquitylated proteins, we used a recently developed affinity purification method to isolate 26S proteasomes from any kind of tissue (7). Equal amounts of the triceps surae from seven animals in each group were pooled and homogenized.…”

Section: Muscle 26s Proteasomes From Aged and Adult Rats Have A Similmentioning

confidence: 99%

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

Altun

Besche

Overkleeft

et al. 2010

Journal of Biological Chemistry

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189

149

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“…Proteasome function is modulated by transiently binding cofactors, the proteasome-interacting proteins (PIPs) (16)(17)(18)(19), which typically are found in substoichiometric amounts in purified proteasomes (20). Of these, ubiquitin C-terminal hydrolase 6 [Ubp6, human ubiquitin-specific protease 14 (Usp14)] is most abundant.…”

Section: Significancementioning

confidence: 99%

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Aufderheide

Beck

Stengel

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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133

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In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediateenergy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.conformational switching | proteolysis | proteostasis | quality control D egradation of proteins that are misfolded, damaged, or no longer needed is an essential element of cellular homeostasis. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is the major pathway for regulated protein degradation (1). Proteins that are processed by the UPS are marked for destruction by polyubiquitin chains, which are recognized as a degradation signal by the 26S proteasome.The 26S proteasome consists of the core particle (CP), which degrades substrates into short peptides, and one or two 19S regulatory particles (RP), which associate with the ends of the cylinder-shaped CP to recruit substrates and prepare them for degradation (2, 3). Although the structure of the CP has been known for more than two decades (4, 5), the molecular architecture of the RP was unraveled by cryo-electron microscope (EM)-based approaches only recently (6-9). It comprises six RP triple A (AAA) ATPases (Rpt), 1-6, and 13 RP non-ATPases (Rpn), 1-3, 5-13, and 15. Similar to AAA-ATPases in prokaryotic ATP-dependent proteases, the Rpts form a hexameric ring that binds to the ends of the CP and is responsible for substrate unfolding and translocation into the CP. Unlike their prokaryotic counterparts, the Rpts are surrounded by non-ATPases. Apart from Rpn1, all Rpns form a cohesive struct...

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“…Our findings that CHIP and VCP bind competitively to mutant SOD1 and that both TPR and U-box domains are necessary for the CHIP's inhibitory effect on SOD1-VCP association could be explained by the assumption that binding of misfolded proteins to 'uncoupling factor' (i.e., VCP) may be an intermediate step in the process of substrate delivery. Interestingly a recent study demonstrated that VCP, which does not have UBL domains, can be isolated together with the 26S proteasomes in the presence of a functional UBL domain derived from HHR23B (human homolog of Rad23) proteins (Besche et al 2009). Our finding that mutant SOD1 also bound to HHR23A protein together with VCP supports our model that VCP Á together with UBL proteins Á mediates the transfer of poly-ubiquitylated mutant SOD1 to the proteolytic machinery.…”

Section: Discussionmentioning

confidence: 99%

“…Like Bag-1, VCP also strongly interacted with mutant SOD1 (A4V and G93A) in the cells ( Figure 3A). Interestingly, an ubiquitin-like domain protein HHR23A (a human homolog of yeast Rad23), which enables the interaction between VCP and 26S proteasomes (Besche et al 2009), also presented in the SOD1 immunoprecipitates ( Figure 3A). In contrast to CHIP, which effectively downregulated G93A SOD1 ( Figure 2B), VCP did not cause any change in the level of mutant SOD1 ( Figure 3B).…”

Section: Bag-1 Stimulates the Interaction Between Chip And Vcpmentioning

confidence: 99%

CHIP promotes the degradation of mutant SOD1 by reducing its interaction with VCP and S6/S6′ subunits of 26S proteasome

Choi

Lee

2010

Animal Cells and Systems

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Previously we showed that CHIP, a co-chaperone of Hsp70 and E3 ubiquitin ligase, can promote the degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis (fALS) via a mechanism not involving SOD1 ubiquitylation. Here we present evidence that CHIP functions in the interaction of mutant SOD1 with 26S proteasomes. Bag-1, a coupling factor between molecular chaperones and the proteasomes, formed a complex with SOD1 in an hsp70-dependent manner but had no direct effect on the degradation of mutant SOD1. Instead, Bag-1 stimulated interaction between CHIP and the proteasome-associated protein VCP (p97), which do not associate normally. Over-expressed CHIP interferedwith the association between mutant SOD1 and VCP. Conversely, the binding of CHIP to mutant SOD1 was inhibited by VCP, implying that the chaperone complex and proteolytic machinery are competing for the common substrates. Finally we observed that mutant SOD1 strongly associated with the 19S complex of proteasomes and CHIP over-expression specifically reduced the interaction between S6/S6? ATPase subunits and mutant SOD1. These results suggest that CHIP, together with ubiquitin-binding proteins such as Bag-1 and VCP, promotes the degradation of mutant SOD1 by facilitating its translocation from ATPase subunits of 19S complex to the 20S core particle.

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Isolation of Mammalian 26S Proteasomes and p97/VCP Complexes Using the Ubiquitin-like Domain from HHR23B Reveals Novel Proteasome-Associated Proteins

Cited by 162 publications

References 70 publications

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

Structural characterization of the interaction of Ubp6 with the 26S proteasome

CHIP promotes the degradation of mutant SOD1 by reducing its interaction with VCP and S6/S6′ subunits of 26S proteasome

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