RNA polymerase (Pol) III transcribes many noncoding RNAs (e.g. tRNAs) important for translational capacity and other functions. Here, we localized Pol III, alternative TFIIIB complexes (BRF1/2) and TFIIIC in HeLa cells, determining the Pol III transcriptome, defining gene classes, and revealing ‘TFIIIC-only’ sites. Pol III localization in other transformed and primary cell lines revealed novel and cell-type specific Pol III loci, and one miRNA. Surprisingly, only a fraction of the in silico-predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Remarkably, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. Taken together, active chromatin appears to gate Pol III accessibility to the genome.

Glut4 exocytosis in adipocytes uses protein machinery that is shared with other regulated secretory processes. Synapsins are phosphoproteins that regulate a `reserve pool' of vesicles clustered behind the active zone in neurons. We found that adipocytes (primary cells and the 3T3-L1 cell line) express synapsin IIb mRNA and protein. Synapsin IIb co-localizes with Glut4 in perinuclear vesicle clusters. To test whether synapsin plays a role in Glut4 traffic, a site 1 phosphorylation mutant (S10A synapsin) was expressed in 3T3-L1 adipocytes. Interestingly, expression of S10A synapsin increased basal cell surface Glut4 almost fourfold (50% maximal insulin effect). Insulin caused a further twofold translocation of Glut4 in these cells. Expression of the N-terminus of S10A synapsin (amino acids 1-118) was sufficient to inhibit basal Glut4 retention. Neither wild-type nor S10D synapsin redistributed Glut4. S10A synapsin did not elevate surface levels of the transferrin receptor in adipocytes or Glut4 in fibroblasts. Therefore, S10A synapsin is inhibiting the specialized process of basal intracellular retention of Glut4 in adipocytes, without affecting general endocytic cycling. While mutant forms of many proteins inhibit Glut4 exocytosis in response to insulin, S10A synapsin is one of only a few that specifically inhibits Glut4 retention in basal adipocytes. These data indicate that the synapsins are important regulators of membrane traffic in many cell types.

Vpr and Vpx are a group of highly related accessory proteins from primate lentiviruses. Despite the high degree of amino acid homology within this group, these proteins can be highly divergent in their functions. In this work, we constructed chimeric and mutant proteins between HIV-1 and SIVagm Vpr in order to better understand the structure-function relationships. We tested these constructs for their abilities to induce G2 arrest in human cells and to degrade agmSAMHD1 and Mus81. We found that the C-terminus of HIV-1 Vpr, when transferred onto SIVagm Vpr, provides the latter with the de novo ability to induce G2 arrest in human cells. We confirmed that HIV-1 Vpr induces degradation of Mus81 although, surprisingly, degradation is independent and genetically separable from Vpr’s ability to induce G2 arrest.

Vpr and Vpx are primate lentivirus proteins that manipulate the cellular CRL4 ubiquitin ligase complex. While Vpr is common to all primate lentiviruses, Vpx is only encoded by HIV-2 and a limited range of SIVs. Although Vpr and Vpx share a high degree of homology they are known to induce markedly different effects in host cell biology through the recruitment of different substrates to CRL4. Here we explore the interaction of HIV-1 Vpr and SIVmac Vpx with the CRL4 substrate receptor DCAF1. Through mutational analysis of DCAF1 we demonstrate that although Vpr and Vpx share a highly similar DCAF1-binding motif, they interact with a different set of residues in DCAF1. In addition, we show that Vpx recruits SAMHD1 through a protein-protein interface that includes interactions of SAMHD1 with both Vpx and DCAF1, as was first suggested in crystallography data by Schwefel et al. (Nature 505:234, 2014).

Background: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is an emergent technology for the treatment of non-compressible torso hemorrhage (NCTH). While aortic occlusion (AO) above the site of hemorrhage provides hemostasis and time for surgical intervention, ischemia-reperfusion injury to the kidneys is a known complication. We aimed to report the incidence and factors associated with acute kidney injury (AKI) following AO in patients with NCTH or in similar porcine models. Methods: We searched Pubmed (MEDLINE), Embase, Scopus, and ProQuest Dissertations & Theses from inception to July 2017. We included original studies of trauma patients with NCTH treated with REBOA, or similar porcine studies that included renal parameters, excluding case reports and case series. After duplicate removal, full texts of studies retrieved via the search strategy were evaluated by two authors. Renal parameters (e.g., creatinine concentration, urine output, histopathology) were extracted. Quality of the evidence and risk of bias were assessed. Results: Twelve out of 2,100 records were included (3 trauma patients, 9 porcine studies). While 1/3 human reports described AO in Zone 1, all swine publications reported Zone 1. All human studies reported renal damage. There were nonetheless inconsistencies in definitions used. Evidence of AKI was reported in 3/9 swine studies.  Conclusions: Consistent reporting of AKI incidence is lacking from human clinical studies of AO in NCTH trauma patients. While comorbidities in trauma patients may contribute to AKI, animal models support the association between AO and AKI. As REBOA is growing in popularity as a therapy for NCTH, further studies determining factors associated with the AKI are needed.