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Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

ABSTRACT: Although cellular proteins conjugated to K48-linked Ub chains are targeted to proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63-ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48-ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48- or K63-ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal-lysosomal pathway.

SUBMITTER: Nathan JA

PROVIDER: S-EPMC3579138 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

Nathan James A JA Kim Hyoung Tae HT Ting Lily L Gygi Steven P SP Goldberg Alfred L AL

The EMBO journal 20130111 4

Although cellular proteins conjugated to K48-linked Ub chains are targeted to proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Usi ...[more]

PMID: 23314748

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Project description:Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.

Dataset Information

Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

Publications

Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

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