To date, neurons have been the primary focus of research on the role of glucocorticoids in the regulation of brain function and pathological behaviors, such as addiction. Astrocytes, which are also ...glucocorticoid-responsive, have been recently implicated in the development of drug abuse, albeit through as yet undefined mechanisms. Here, using a spectrum of tools (whole-transcriptome profiling, viral-mediated RNA interference in vitro and in vivo, behavioral pharmacology and electrophysiology), we demonstrate that astrocytes in the nucleus accumbens (NAc) are an important locus of glucocorticoid receptor (GR)-dependent transcriptional changes that regulate rewarding effects of morphine. Specifically, we show that targeted knockdown of the GR in the NAc astrocytes enhanced conditioned responses to morphine, with a concomitant inhibition of morphine-induced neuronal excitability and plasticity. Interestingly, GR knockdown did not influence sensitivity to cocaine. Further analyses revealed GR-dependent regulation of astroglial metabolism. Notably, GR knockdown inhibited induced by glucocorticoids lactate release in astrocytes. Finally, lactate administration outbalanced conditioned responses to morphine in astroglial GR knockdown mice. These findings demonstrate a role of GR-dependent regulation of astrocytic metabolism in the NAc and a key role of GR-expressing astrocytes in opioid reward processing.
Effective delivery of services for the public good involves a multiplicity of organizations and actors, including those from the public, nonprofit, and private sectors. In some cases, service ...delivery is accomplished using programs that directly engage volunteers, including key public services like community-based and nonprofit volunteer fire departments. Volunteers in fire departments—often highly engaged volunteers with specialized training—provide vital services for a substantial portion of the United States, allowing local governments to realize considerable cost savings. Thus, issues of volunteer retention are a critical challenge for fire departments. Existing research has addressed issues of retention in a variety of settings; we argue that the challenging and particular context of fire departments is worthy of focused research. This article is an exploratory study of the predictors of voluntary firefighter retention in the Commonwealth of Pennsylvania. We use data from a web-based survey of volunteer firefighters to examine the factors that influence volunteer retention, focusing specifically on volunteer management practices and broader job-related concepts. Results indicate that volunteer training, performance management, job satisfaction, and organizational commitment influence both short- and mid-term intent to remain, indicating that management practices and programs, as well as other contextual factors that shape satisfaction and commitment, are important in retaining volunteers.
Natural products have demonstrated utility in the clinic and can also act as probes to understand complex cellular pathways. Sanglifehrin A (SFA) is a mixed polyketide and non-ribosomal peptide ...synthase natural product with sub-nano-molar affinity for its receptor cyclophilin A (PPIA). It has been shown to behave in vitro as an immune suppressant. Here, we identify inosine-5′-monophosphate dehydrogenase 2 (IMPDH2) as an intracellular target of the PPIA-SFA binary complex. The formation of this ternary complex does not inhibit the enzymatic activity of IMPDH2. Rather, ternary complex formation modulates cell growth through interaction with the cystathionine-β-synthase (CBS) domain of IMPDH2. We further demonstrate that the SFA complex is highly isoform selective for IMPDH2 (versus IMPDH1). This work reveals a role for the CBS domains of IMPDH2 in cellular proliferation, suggesting a more complex role than previously suspected for IMPDH2 in T cell activation and proliferation.
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•SFA engages IMPDH2 in a PPIA-dependent manner•PPIA-SFA interaction with IMPDH is highly isoform specific•PPIA-SFA complex binds the CBS domains of IMPDH2•CBS domains of IMPDH2 are implicated in cellular proliferation
Pua et al. identify IMPDH2 as an intracellular target of the PPIA-SFA complex and show that the CBS domains of IMPDH2 are required for cellular proliferation.
Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology ...for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.
Vertebrates express two enzymes for activation of ubiquitin—UBA1, which is responsible for activation of the vast majority of E2 conjugating enzymes, and UBA6, which uses the dedicated E2, USE1. ...However, targets and E3s for UBA6-USE1 are unknown. Here, we demonstrate that UBA6-USE1 functions with the UBR1–3 subfamily of N-recognin E3s to degrade the N-end rule substrates RGS4, RGS5, and Arg (R)-GFP. This pathway functions in the cytoplasm in parallel with the UBA1-UBE2A/B-UBR2 cascade, which promotes turnover of nuclear RGS4/5 proteins and an apparently phenotypically distinct pool of cytoplasmic RGS4/5. UBR2 promotes Lys48 (K48)-specific ubiquitin discharge from, and RGS4 ubiquitylation by, both USE1 and UBE2A in vitro. This work provides insight into the machinery employed by the UBA6-USE1 cascade to promote protein turnover and suggests that the UBA6 and UBA1 pathways can function in parallel with the same E3 to degrade the same targets in a spatially distinct manner.
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► USE1 interacts with members of the N-recognin family of ubiquitin ligases ► The RING domain of UBR2 promotes Lys48-specific ubiquitin discharge from USE1 ► UBR2 promotes Lys48-specific ubiquitin conjugation of RGS4 in vitro ► USE1 and UBE2A/B, a second E2 for UBR2, promote RGS4 turnover in vivo
UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to ...contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.
An essential step in the pathogenesis of human papillomavirus (HPV)-associated cancers is the dysregulated expression of the viral oncogenes. The papillomavirus E2 protein can silence the long ...control region (LCR) promoter that controls viral E6 and E7 oncogene expression. The mechanisms by which E2 represses oncogene expression and the cellular factors through which E2 mediates this silencing are largely unknown. We conducted an unbiased, genome-wide siRNA screen and series of secondary screens that identified 96 cellular genes that contribute to the repression of the HPV LCR. In addition to confirming a role for the E2-binding bromodomain protein Brd4 in E2-mediated silencing, we identified a number of genes that have not previously been implicated in E2 repression, including the demethylase JARID1C/SMCX as well as EP400, a component of the NuA4/TIP60 histone acetyltransferase complex. Each of these genes contributes independently and additively to E2-mediated silencing, indicating that E2 functions through several distinct cellular complexes to repress E6 and E7 expression.
von Willebrand factor (vWF) mediates platelet adhesion and thrombus formation via its interaction with the platelet receptor glycoprotein (GP)Ibα. We have analyzed two A1A2A3 tri-domain proteins to ...demonstrate that the amino acid sequence, Gln1238-Glu1260, in the N-terminal flanking region of the A1 domain, together with the association between the A domains, modulates vWF-GPIbα binding and platelet activation under shear stress. Using circular dichroism spectroscopy and differential scanning calorimetry, we have described that sequence Gln1238-Glu1260 stabilizes the structural conformation of the A1A2A3 tri-domain complex. The structural stabilization imparted by this particular region inhibits the binding capacity of the tri-domain protein for GPIbα. Deletion of this region causes a conformational change in the A1 domain that increases binding to GPIbα. Only the truncated protein was capable of effectively blocking ristocetin-induced platelet agglutination. To determine the capacity of activating platelets via the interaction with GPIbα, whole blood was incubated with the N-terminal region truncated or intact tri-A domain protein prior to perfusion over a fibrin(ogen)-coated surface. At a high shear rate of 1,500 s−1, platelets from blood containing the truncated protein rapidly bound, covering >90% of the fibrin(ogen) surface area, whereas the intact tri-A domain protein induced platelets to bind <10%. The results obtained in this study ascertain the relevant role of the structural association between the N-terminal flanking region of the A1 domain (amino acids Gln1238-Glu1260) and the A1A2A3 domain complex in preventing vWF to bind spontaneously to GPIbα in solution under high shear forces.
Background: A conformational change in A1 domain of vWF is required for GPIbα binding.
Results: N-terminal flanking region of the A1 domain inhibits binding to GPIbα, stabilizes the A1A2A3 structure, and prevents platelet activation.
Conclusion: Coupling between the N-terminal flanking region of the A1 domain and the A1A2A3 complex inhibits vWF-GPIbα interaction.
Significance: This autoinhibitory mechanism prevents spontaneous thrombosis in circulation.
Visual imagery is a fundamental element of our communication systems. Unlike spoken or written words that deliver meaning one word at a time, images convey multiple messages instantly with powerful ...impact. This study explores the content embedded in the social media images used by nonprofit police foundations in the United States through archeological visual analysis. Findings reveal that public service organizations navigate curation choices when selecting images for social media accounts, such as how best to depict the mission of the organization. The prioritization of image curation is imperative, especially with the growth of social media as a space for promoting encounters with, not just information distribution to, citizens. In the public service, the importance of image curation lies in the potential to reinforce the work of organizations, but the risk is miscommunication with consequences for public trust.
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