The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA domain is responsible for this activity. Two other proteins containing this motif, the fission-yeast homologues of Rad23 and Dsk2, are also shown to bind multi-ubiquitin chains via their UBA domains. These two proteins are implicated, along with the fission-yeast Pus1(S5a/Rpn10) subunit of the 26 S proteasome, in the recognition and turnover of substrates by this proteolytic complex.
Ubiquitin-mediated processes orchestrate critical DNA-damage signaling and repair pathways. We identify human DVC1 (C1orf124; Spartan) as a cell cycle-regulated anaphase-promoting complex (APC) substrate that accumulates at stalled replication forks. DVC1 recruitment to sites of replication stress requires its ubiquitin-binding UBZ domain and PCNA-binding PIP box motif but is independent of RAD18-mediated PCNA monoubiquitylation. Via a conserved SHP box, DVC1 recruits the ubiquitin-selective chaperone p97 to blocked replication forks, which may facilitate p97-dependent removal of translesion synthesis (TLS) DNA polymerase η (Pol η) from monoubiquitylated PCNA. DVC1 knockdown enhances UV light-induced mutagenesis, and depletion of human DVC1 or the Caenorhabditis elegans ortholog DVC-1 causes hypersensitivity to replication stress-inducing agents. Our findings establish DVC1 as a DNA damage-targeting p97 adaptor that protects cells from deleterious consequences of replication blocks and suggest an important role of p97 in ubiquitin-dependent regulation of TLS.
The rapid decrease in DNA sequencing cost is revolutionizing medicine and science. In medicine, genome sequencing has revealed millions of missense variants that change protein sequences, yet we only understand the molecular and phenotypic consequences of a small fraction. Within protein science, high-throughput deep mutational scanning experiments enable us to probe thousands of variants in a single, multiplexed experiment. We review efforts that bring together these topics via experimental and computational approaches to determine the consequences of missense variants in proteins. We focus on the role of changes in protein stability as a driver for disease, and how experiments, biophysical models and computation are providing a framework for understanding and predicting how changes in protein sequence affect cellular protein stability.
Accurate methods to assess the pathogenicity of mutations are needed to fully leverage the possibilities of genome sequencing in diagnosis. Current data-driven and bioinformatics approaches are, however, limited by the large number of new variations found in each newly sequenced genome, and often do not provide direct mechanistic insight. Here we demonstrate, for the first time, that saturation mutagenesis, biophysical modeling and co-variation analysis, performed in silico, can predict the abundance, metabolic stability, and function of proteins inside living cells. As a model system, we selected the human mismatch repair protein, MSH2, where missense variants are known to cause the hereditary cancer predisposition disease, known as Lynch syndrome. We show that the majority of disease-causing MSH2 mutations give rise to folding defects and proteasome-dependent degradation rather than inherent loss of function, and accordingly our in silico modeling data accurately identifies disease-causing mutations and outperforms the traditionally used genetic disease predictors. Thus, in conclusion, in silico biophysical modeling should be considered for making genotype-phenotype predictions and for diagnosis of Lynch syndrome, and perhaps other hereditary diseases.
SummaryThe ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48. Atomic resolution data show that Dss1 is disordered and binds ubiquitin by binding sites characterized by acidic and hydrophobic residues. The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions. Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins.
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