Human single-nucleotide polymorphisms alter p53 sequence-specific binding at gene regulatory elements

p53 coordinates the expression of an intricate network of genes in response to stress signals. Sequence-specific DNA binding is essential for p53-mediated tumor suppression. We evaluated the impact of single-nucleotide polymorphisms (SNPs) in p53 response elements (p53RE) on DNA binding and gene expression in response to DNA damage. Using a bioinformatics approach based on incorporating p53 binding strength into a position weight matrix, we selected 32 SNPs in putative and validated p53REs. The microsphere assay for protein–DNA binding (MAPD) and allele-specific expression analysis was employed to assess the impact of SNPs on p53-DNA binding and gene expression, respectively. Comparing activated p53 binding in nuclear extracts from doxorubicin- or ionizing radiation (IR)-treated human cells, we observed little difference in binding profiles. Significant p53 binding was observed for most polymorphic REs and several displayed binding comparable to the p21 RE. SNP alleles predicted to lower p53 binding indeed reduced binding in 25 of the 32 sequences. Chromatin immunoprecipitation-sequencing in lymphoblastoid cells confirmed p53 binding to seven polymorphic p53 REs in response to doxorubicin. In addition, five polymorphisms were associated with altered gene expression following doxorubicin treatment. Our findings demonstrate an effective strategy to identify and evaluate SNPs that may alter p53-mediated stress responses

Kaposi's Sarcoma Herpesvirus microRNAs Target Caspase 3 and Regulate Apoptosis

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis

The Interferon Response Inhibits HIV Particle Production by Induction of TRIM22

Treatment of human cells with Type 1 interferons restricts HIV replication. Here we report that the tripartite motif protein TRIM22 is a key mediator. We used transcriptional profiling to identify cellular genes that were induced by interferon treatment and identified TRIM22 as one of the most strongly up-regulated genes. We confirmed, as in previous studies, that TRIM22 over-expression inhibited HIV replication. To assess the role of TRIM22 expressed under natural inducing conditions, we compared the effects of interferon in cells depleted for TRIM22 using RNAi and found that HIV particle release was significantly increased in the knockdown, implying that TRIM22 acts as a natural antiviral effector. Further studies showed that TRIM22 inhibited budding of virus-like particles containing Gag only, indicating that Gag was the target of TRIM22. TRIM22 did not block the release of MLV or EIAV Gag particles. Inhibition was associated with diffuse cytoplasmic staining of HIV Gag rather than accumulation at the plasma membrane, suggesting TRIM22 disrupts proper trafficking. Mutational analyses of TRIM22 showed that the catalytic amino acids Cys15 and Cys18 of the RING domain are required for TRIM22 antiviral activity. These data disclose a pathway by which Type 1 interferons obstruct HIV replication

Venous gas embolism as a predictive tool for improving CNS decompression safety

A key process in the pathophysiological steps leading to decompression sickness (DCS) is the formation of inert gas bubbles. The adverse effects of decompression are still not fully understood, but it seems reasonable to suggest that the formation of venous gas emboli (VGE) and their effects on the endothelium may be the central mechanism leading to central nervous system (CNS) damage. Hence, VGE might also have impact on the long-term health effects of diving. In the present review, we highlight the findings from our laboratory related to the hypothesis that VGE formation is the main mechanism behind serious decompression injuries. In recent studies, we have determined the impact of VGE on endothelial function in both laboratory animals and in humans. We observed that the damage to the endothelium due to VGE was dose dependent, and that the amount of VGE can be affected both by aerobic exercise and exogenous nitric oxide (NO) intervention prior to a dive. We observed that NO reduced VGE during decompression, and pharmacological blocking of NO production increased VGE formation following a dive. The importance of micro-nuclei for the formation of VGE and how it can be possible to manipulate the formation of VGE are discussed together with the effects of VGE on the organism. In the last part of the review we introduce our thoughts for the future, and how the enigma of DCS should be approached

Selective autophagy degrades DICER and AGO2 and regulates miRNA activity.

MicroRNAs (miRNAs) form a class of short RNAs (∼ 21 nucleotides) that post-transcriptionally regulate partially complementary messenger RNAs. Each miRNA may target tens to hundreds of transcripts to control key biological processes. Although the biochemical reactions underpinning miRNA biogenesis and activity are relatively well defined and the importance of their homeostasis is increasingly evident, the processes underlying regulation of the miRNA pathway in vivo are still largely elusive. Autophagy, a degradative process in which cytoplasmic material is targeted into double-membrane vacuoles, is recognized to critically contribute to cellular homeostasis. Here, we show that the miRNA-processing enzyme, DICER (also known as DICER1), and the main miRNA effector, AGO2 (also known as eukaryotic translation initiation factor 2C, 2 (EIF2C2)), are targeted for degradation as miRNA-free entities by the selective autophagy receptor NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2)). Autophagy establishes a checkpoint required for continued loading of miRNA into AGO2; accordingly, NDP52 and autophagy are required for homeostasis and activity of the tested miRNAs. Autophagy also engages post-transcriptional regulation of the DICER mRNA, underscoring the importance of fine-tuned regulation of the miRNA pathway. These findings have implications for human diseases linked to misregulated autophagy, DICER- and miRNA-levels, including cancer

TRIM5α and TRIM22 are differentially regulated according to HIV-1 infection phase and compartment.

CAPRISA, 2014.The antiviral role of TRIM E3 ligases in vivo is not fully understood. To test the hypothesis that TRIM5α and TRIM22 have differential transcriptional regulation and distinct anti-HIV roles according to infection phase and compartment, we measured TRIM5α, TRIM22, and type I interferon (IFN-I)-inducible myxovirus resistance protein A (MxA) levels in peripheral blood mononuclear cells (PBMCs) during primary and chronic HIV-1 infection, with chronic infection samples being matched PBMCs and central nervous system (CNS)-derived cells. Associations with biomarkers of disease progression were explored. The impact of IFN-I, select proinflammatory cytokines, and HIV on TRIM E3 ligase-specific expression was investigated. PBMCs from individuals with primary and chronic HIV-1 infection had significantly higher levels of MxA and TRIM22 than did PBMCs from HIV-1-negative individuals (P < 0.05 for all comparisons). PBMCs from chronic infection had lower levels of TRIM5α than did PBMCs from primary infection or HIV-1-uninfected PBMCs (P = 0.0001 for both). In matched CNS-derived samples and PBMCs, higher levels of MxA (P = 0.001) and TRIM5α (P = 0.0001) in the CNS were noted. There was a negative correlation between TRIM22 levels in PBMCs and plasma viral load (r = -0.40; P = 0.04). In vitro, IFN-I and, rarely, proinflammatory cytokines induced TRIM5α and TRIM22 in a cell type-dependent manner, and the knockdown of either protein in CD4(+) lymphocytes resulted in increased HIV-1 infection. These data suggest that there are infection-phase-specific and anatomically compartmentalized differences in TRIM5α and TRIM22 regulation involving primarily IFN-I and specific cell types and indicate subtle differences in the antiviral roles and transcriptional regulation of TRIM E3 ligases in vivo