Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis

The pathophysiology of restricted repetitive behavior

Restricted, repetitive behaviors (RRBs) are heterogeneous ranging from stereotypic body movements to rituals to restricted interests. RRBs are most strongly associated with autism but occur in a number of other clinical disorders as well as in typical development. There does not seem to be a category of RRB that is unique or specific to autism and RRB does not seem to be robustly correlated with specific cognitive, sensory or motor abnormalities in autism. Despite its clinical significance, little is known about the pathophysiology of RRB. Both clinical and animal models studies link repetitive behaviors to genetic mutations and a number of specific genetic syndromes have RRBs as part of the clinical phenotype. Genetic risk factors may interact with experiential factors resulting in the extremes in repetitive behavior phenotypic expression that characterize autism. Few studies of individuals with autism have correlated MRI findings and RRBs and no attempt has been made to associate RRB and post-mortem tissue findings. Available clinical and animal models data indicate functional and structural alterations in cortical-basal ganglia circuitry in the expression of RRB, however. Our own studies point to reduced activity of the indirect basal ganglia pathway being associated with high levels of repetitive behavior in an animal model. These findings, if generalizable, suggest specific therapeutic targets. These, and perhaps other, perturbations to cortical basal ganglia circuitry are mediated by specific molecular mechanisms (e.g., altered gene expression) that result in long-term, experience-dependent neuroadaptations that initiate and maintain repetitive behavior. A great deal more research is needed to uncover such mechanisms. Work in areas such as substance abuse, OCD, Tourette syndrome, Parkinson’s disease, and dementias promise to provide findings critical for identifying neurobiological mechanisms relevant to RRB in autism. Moreover, basic research in areas such as birdsong, habit formation, and procedural learning may provide additional, much needed clues. Understanding the pathophysioloy of repetitive behavior will be critical to identifying novel therapeutic targets and strategies for individuals with autism

The trans-ancestral genomic architecture of glycemic traits

Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P < 5 x 10(-8)), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution. A trans-ancestry meta-analysis of GWAS of glycemic traits in up to 281,416 individuals identifies 99 novel loci, of which one quarter was found due to the multi-ancestry approach, which also improves fine-mapping of credible variant sets.Peer reviewe

Ubp2 Regulates Rsp5 Ubiquitination Activity <i>In Vivo</i> and <i>In Vitro</i>

<div><p>The yeast HECT-family E3 ubiquitin ligase Rsp5 has been implicated in diverse cell functions. Previously, we and others <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075372#pone.0075372-Kee1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075372#pone.0075372-Lam1" target="_blank">[2]</a> reported the physical and functional interaction of Rsp5 with the deubiquitinating enzyme Ubp2, and the <u>u</u>biquitin <u>a</u>ssociated (UBA) domain-containing cofactor Rup1. To investigate the mechanism and significance of the Rsp5-Rup1-Ubp2 complex, we examined Rsp5 ubiquitination status in the presence or absence of these cofactors. We found that, similar to its mammalian homologues, Rsp5 is auto-ubiquitinated <i>in vivo</i>. Association with a substrate or Rup1 increased Rsp5 self-ubiquitination, whereas Ubp2 efficiently deubiquitinates Rsp5 <i>in vivo</i> and <i>in vitro.</i> The data reported here imply an auto-modulatory mechanism of Rsp5 regulation common to other E3 ligases.</p></div

Rup1 stimulates Rsp5 auto-ubiquitination.

<p>Western blot analysis of an <i>in vitro</i> Rsp5 ubiquitination reaction with CTD (+CTD lanes) or lacking CTD in the presence of Rup1-TAP (+Rup1 lanes) purified from a strain lacking <i>UBP2</i>. Reactions were incubated from 5 minutes to 4 hours as indicated and stopped by the addition of sample loading buffer. Ubiquitinated and unmodified forms of GST-tagged Rsp5 were detected with anti-GST antibody.</p

Rsp5 is stably ubiquitinated in the absence of Ubp2.

<p>(A) Western blot showing ubiquitinated and unmodified forms of Rsp5 <i>in vivo</i>. A plasmid expressing epitope-tagged Rsp5 (pHA-Rsp5) was transformed into wildtype and <i>ubp2</i>Δ cells. Cells were lysed in native buffer, and Rsp5 immunoprecipitated with anti-HA antibodies. After transfer to nitrocellulose, Rsp5 species were visualized using anti-ubiquitin (top panel) and anti-HA antibodies (middle; longer exposure at bottom). Whole cell extract from cells lacking plasmid (WCE) and immunoprecipitates from these same cells (WT) were run as positive and negative controls, respectively. (B) Experiment performed as in (A), except with an increased exposure time in order to visualize lower mobility Ub-HA-Rsp5 species. Accumulation of ubiquitinated HA-Rsp5 is evident in a strain bearing a catalytically inactive mutant allele in <i>UBP2</i> (<i>ubp2 C745S</i>). (C) Rsp5 is auto-ubiquitinated. Plasmids expressing either wildtype Rsp5 (pHA-Rsp5 WT) or a conditional catalytic mutant of Rsp5 (pHA-rsp5-1) were transformed into haploid yeast lacking <i>UBP2</i> alone or both <i>UBP2</i> and a fully functional copy of <i>RSP5</i> (<i>rsp5-1</i>). Prior to analysis, the strains were grown overnight, diluted in fresh media, and incubated at the non-permissive temperature (37°C) to inactivate Rsp5. Cell lysates were immunoprecipitated with anti-HA antibodies, and the blot first probed with anti-ubiquitin, followed by extended wash steps to rinse off residual antibody, and finally Rsp5 was detected with anti-HA antibodies. A darker exposure of the anti-HA blot is shown to highlight the lack of low-mobility (multi/poly-ubiquitinated) forms of Rsp5-1.</p

Substrate-induced Rsp5 auto-ubiquitination.

<p>Western blot analysis of an <i>in vitro</i> Rsp5 ubiquitination reaction in the (A) presence (+CTD lanes) or (B) absence of substrate (-CTD lanes). Reactions (E1, E2, Rsp5, ATP, buffer, Ub,+/− CTD) were incubated from 15 to 30 minutes as indicated, and stopped by the addition of sample loading buffer. Both GST-Rsp5 and GST-CTD were detected with anti-GST antibody, and Rsp5 (unmodified and modified forms) detected on a separately run blot with anti-Rsp5 antibody. Asterisks in (B, upper panel) denote degradation products of Rsp5. These bands are not present in (A, upper panel), as they are obscured by the CTD band. Ubiquitinated and unmodified forms of Rsp5 and CTD are indicated.</p