No abstract

The ability of ubiquitin to form up to eight different polyubiquitin chain linkages generates complexity within the ubiquitin proteasome system, and accounts for the diverse roles of ubiquitination within the cell. Understanding how each type of ubiquitin linkage is correctly interpreted by ubiquitin binding proteins provides important insights into the link between chain recognition and cellular fate. A major function of ubiquitination is to signal degradation of intracellular proteins by the 26S proteasome. Lysine-48 (K48) linked polyubiquitin chains are well established as the canonical signal for proteasomal degradation, but recent studies show a role for other ubiquitin linked chains in facilitating degradation by the 26S proteasome. Here, we review how different types of polyubiquitin linkage bind to ubiquitin receptors on the 26S proteasome, how they signal degradation and discuss the implications of ubiquitin chain linkage in regulating protein breakdown by the proteasome.

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

SummaryHypoxia-inducible transcription factors (HIFs) control adaptation to low oxygen environments by activating genes involved in metabolism, angiogenesis, and redox homeostasis. The finding that HIFs are also regulated by small molecule metabolites highlights the need to understand the complexity of their cellular regulation. Here we use a forward genetic screen in near-haploid human cells to identify genes that stabilize HIFs under aerobic conditions. We identify two mitochondrial genes, oxoglutarate dehydrogenase (OGDH) and lipoic acid synthase (LIAS), which when mutated stabilize HIF1α in a non-hydroxylated form. Disruption of OGDH complex activity in OGDH or LIAS mutants promotes L-2-hydroxyglutarate formation, which inhibits the activity of the HIFα prolyl hydroxylases (PHDs) and TET 2-oxoglutarate dependent dioxygenases. We also find that PHD activity is decreased in patients with homozygous germline mutations in lipoic acid synthesis, leading to HIF1 activation. Thus, mutations affecting OGDHC activity may have broad implications for epigenetic regulation and tumorigenesis.

The US11 gene product of human cytomegalovirus promotes viral immune evasion by hijacking the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. US11 initiates dislocation of newly translocated MHC I from the ER to the cytosol for proteasome-mediated degradation. Despite the critical role for ubiquitin in this degradation pathway, the responsible E3 ligase is unknown. In a forward genetic screen for host ERAD components hijacked by US11 in near-haploid KBM7 cells, we identified TMEM129, an uncharacterized polytopic membrane protein. TMEM129 is essential and rate-limiting for US11-mediated MHC-I degradation and acts as a novel ER resident E3 ubiquitin ligase. TMEM129 contains an unusual cysteine-only RING with intrinsic E3 ligase activity and is recruited to US11 via Derlin-1. Together with its E2 conjugase Ube2J2, TMEM129 is responsible for the ubiquitination, dislocation, and subsequent degradation of US11-associated MHC-I. US11 engages two degradation pathways: a Derlin-1/TMEM129-dependent pathway required for MHC-I degradation and a SEL1L/HRD1-dependent pathway required for "free" US11 degradation. Our data show that TMEM129 is a novel ERAD E3 ligase and the central component of a novel mammalian ERAD complex. P roteins inserted into the endoplasmic reticulum (ER) must fold and acquire their native state before further trafficking through the secretory pathway (1, 2). To avoid the toxicity associated with misfolded gene products, all proteins must pass the ER quality-control checkpoint. Misfolded proteins are rejected and dislocated across the ER-membrane for cytosolic proteasome degradation in a process known as ER-associated degradation (ERAD). ERAD degrades misfolded and unassembled proteins and regulates turnover of ER-resident proteins (3).Ubiquitination of the protein substrate provides a critical step in protein dislocation (4). The RING family constitutes the largest family of E3 ligases, including those involved in ERAD (5). The RING domain creates a binding platform for the E2 conjugase and consists of two zinc atoms coordinated in a cross-brace motif via interspersed cysteine (C) and histidine (H) residues in a C3HC4, C3H2C3, or C4HC3 conformation (5). Most ERAD E3 ligases are integral membrane proteins; they form the core of the ERAD machinery, nucleating functionally distinct ERAD complexes. The mammalian system is more complex than yeast and has undergone an expansion of the ERAD E3 ligase family, with the Hrd1p homologs Hrd1 and Gp78, the Doa10p homolog MARCH6, RNF5, TRC8, and CHIP (3, 4).Many pathogens appropriate the ubiquitin-proteasome system, particularly to degrade components of the host immune system (6, 7). The human cytomegalovirus (HCMV) US2 and US11 gene products have been instrumental in studies of the mammalian ERAD system. These viral proteins hijack separate components of the ERAD system to degrade MHC-I, thus preventing cytotoxic T lymphocyte recognition of infected cells (8,9). US2 appropriates the TRC8 E3 ubiquitin ligase (10), whereas dislocation induced by US1...