RNAi screening for kinases and phosphatases identifies FoxO regulators

Forkhead box class O (FoxO) transcription factors are key regulators of growth, metabolism, life span, and stress resistance. FoxOs integrate signals from different pathways and guide the cellular response to varying energy and stress conditions. FoxOs are modulated by several signaling pathways, e.g., the insulin-TOR signaling pathway and the stress induced JNK signaling pathway. Here, we report a genome wide RNAi screen of kinases and phosphatases aiming to find regulators of dFoxO activity in Drosophila S2 cells. By using a combination of transcriptional activity and localization assays we identified several enzymes that modulate dFoxO transcriptional activity, intracellular localization and/or protein stability. Importantly, several currently known dFoxO regulators were found in the screening, confirming the validity of our approach. In addition, several interesting new regulators were identified, including protein kinase C and glycogen synthase kinase 3β, two proteins with important roles in insulin signaling. Furthermore, several mammalian orthologs of the proteins identified in Drosophila also regulate FOXO activity in mammalian cells. Our results contribute to a comprehensive understanding of FoxO regulatory processes

Williams-Beuren syndrome TRIM50 encodes an E3 ubiquitin ligase.

Williams-Beuren syndrome (WBS) is a neurodevelopmental and multisystemic disease that results from hemizygosity of approximately 25 genes mapping to chromosomal region 7q11.23. We report here the preliminary description of eight novel genes mapping within the WBS critical region and/or its syntenic mouse region. Three of these genes, TRIM50, TRIM73 and TRIM74, belong to the TRIpartite motif gene family, members of which were shown to be associated to several human genetic diseases. We describe the preliminary functional characterization of these genes and show that Trim50 encodes an E3 ubiquitin ligase, opening the interesting hypothesis that the ubiquitin-mediated proteasome pathway might be involved in the WBS phenotype

Role of TRIM5α RING Domain E3 Ubiquitin Ligase Activity in Capsid Disassembly, Reverse Transcription Blockade, and Restriction of Simian Immunodeficiency Virus▿†

The mammalian tripartite motif protein, TRIM5α, recognizes retroviral capsids entering the cytoplasm and blocks virus infection. Depending on the particular TRIM5α protein and retrovirus, complete disruption of the TRIM5α RING domain decreases virus-restricting activity to various degrees. TRIM5α exhibits RING domain-dependent E3 ubiquitin ligase activity, but the specific role of this activity in viral restriction is unknown. We created a panel of African green monkey TRIM5α (TRIM5αAGM) mutants, many of which are specifically altered in RING domain E3 ubiquitin ligase function, and characterized the phenotypes of these mutants with respect to restriction of simian and human immunodeficiency viruses (SIVmac and HIV-1, respectively). TRIM5αAGM ubiquitin ligase activity was essential for both the accelerated disassembly of SIVmac capsids and the disruption of reverse transcription. The levels of SIVmac particulate capsids in the cytosol of target cells expressing the TRIM5α variants strongly correlated with the levels of viral late reverse transcripts. RING-mediated ubiquitylation and B30.2(SPRY) domain-determined capsid binding independently contributed to the potency of SIVmac restriction by TRIM5αAGM. In contrast, TRIM5α proteins attenuated in RING ubiquitin ligase function still accelerated HIV-1 capsid disassembly, inhibited reverse transcription, and blocked infection. Replacement of the helix-4/5 loop in the SIVmac capsid with the corresponding region of the HIV-1 capsid diminished the dependence of restriction on TRIM5α RING function. Thus, ubiquitylation mediated by the RING domain of TRIM5αAGM is essential for blocking SIVmac infection at the stage of capsid uncoating

TRIM E3 Ubiquitin Ligases in Rare Genetic Disorders

The TRIM family comprises proteins characterized by the presence of the tripartite motif composed of a RING domain, one or two B-box domains and a coiled-coil region. The TRIM shared domain structure underscores a common biochemical function as E3 ligase within the ubiquitination cascade. The TRIM proteins represent one of the largest E3 ligase families counting in human more than 70 members. These proteins are implicated in a plethora of cellular processes such as apoptosis, cell cycle regulation, muscular physiology, and innate immune response. Consistently, their alteration results in several pathological conditions emphasizing their medical relevance. Here, the genetic and pathogenetic mechanisms of rare disorders directly caused by mutations in TRIM genes will be reviewed. These diseases fall into different pathological areas, from malformation birth defects due to developmental abnormalities, to neurological disorders and progressive teenage neuromuscular disorders. In many instances, TRIM E3 ligases act on several substrates thus exerting pleiotropic activities: the need of unraveling disease-specific TRIM pathways for a precise targeting therapy avoiding dramatic side effects will be discussed

Anti-HMGB1 Neutralizing Antibody Ameliorates Gut Barrier Dysfunction and Improves Survival after Hemorrhagic Shock

Intestinal barrier dysfunction occurs following hemorrhagic shock and resuscitation (HS/R). High-mobility group B1 (HMGB1) has been shown to increase the permeability of Caco-2 human enterocyte-like epithelial monolayers in vitro. In this study, we found that serum concentrations of HMGB1 were higher in blood samples obtained from 25 trauma victims with hemorrhagic shock than in 9 normal volunteers. We also studied whether treatment with anti-HMGB1 antibody can ameliorate HS/R-induced gut barrier dysfunction in mice. Animals were shocked by withdrawal of blood to maintain mean arterial pressure at 25 to 30 mmHg for 2 h. After resuscitation with shed blood plus Ringer’s lactate solution, the mice were treated with either anti-HMGB1 antibody or nonimmune rabbit IgG. Serum HMGB1 concentrations were significantly higher in trauma victims than control mice. Treatment with anti-HMGB1 antibody improved survival at 24 h and ameliorated the development of ileal mucosal hyperpermeability to FITC-labeled dextran. At 24 h after HS/R, treatment with anti-HMGB1 antibody decreased bacterial translocation to mesenteric lymph nodes and was associated with lower circulating concentrations of IL-6 and IL-10. These data support the notion that HMGB1 is a mediator of HS/R-induced gut barrier dysfunction and suggest that anti-HMGB1 antibodies warrant further evaluation as a therapeutic to ameliorate the morbidity of HS/R in trauma patients