HIV-1 is dependent on its immature lattice to recruit IP6 for mature capsid assembly

HIV-1 Gag metamorphoses inside each virion, from an immature lattice that forms during viral production to a mature capsid that drives infection. Here we show that the immature lattice is required to concentrate the cellular metabolite inositol hexakisphosphate (IP6) into virions to catalyze mature capsid assembly. Disabling the ability of HIV-1 to enrich IP6 does not prevent immature lattice formation or production of the virus. However, without sufficient IP6 molecules inside each virion, HIV-1 can no longer build a stable capsid and fails to become infectious. IP6 cannot be replaced by other inositol phosphate (IP) molecules, as substitution with other IPs profoundly slows mature assembly kinetics and results in virions with gross morphological defects. Our results demonstrate that while HIV-1 can become independent of IP6 for immature assembly, it remains dependent upon the metabolite for mature capsid formation

The gut microbiome in dogs with congestive heart failure: a pilot study

Compromised gut health and dysbiosis in people with heart failure has received a great deal of attention over the last decade. Whether dogs with heart failure have a similar dysbiosis pattern to what is described in people is currently unknown. We hypothesised that dogs with congestive heart failure have quantifiable dysbiosis compared to healthy dogs that are similar in sex and age. A total of 50 dogs (15 healthy dogs and 35 dogs with congestive heart failure) were prospectively recruited, and their faecal gut microbiome was assessed using 16S rRNA sequencing (Illumina MiSeq platform). There was no significant change in the microbial diversity and richness in dogs with congestive heart failure. However, there was an increase in abundance of Proteobacteria in the congestive heart failure group (p = 0.014), particularly due to an increase in the family Enterobacteriaceae (p = 0.002) and Escherichia coli (p = 0.033). We conclude that dogs with congestive heart failure have dysbiosis, and we show additional trends in our data suggesting that dogs may have a similar pattern to that described in people. The results of this study provide useful preliminary information and raise the possibility that dogs represent a clinically relevant animal model of dysbiosis in people with heart failure

Acute and rapid degradation of endogenous proteins by Trim-Away.

Protein depletion is a key approach to understanding the functions of a protein in a biological system. We recently developed the Trim-Away approach in order to rapidly degrade endogenous proteins without prior modification. Trim-Away is based on the ubiquitin ligase and Fc receptor TRIM21, which recognizes antibody-bound proteins and targets them for degradation by the proteasome. In a typical Trim-Away experiment, protein degradation is achieved in three steps: first, introduction of an antibody against the target protein; second, recruitment of endogenous or exogenous/overexpressed TRIM21 to the antibody-bound target protein; and third, proteasome-mediated degradation of the target protein, antibody and TRIM21 complex. Protein degradation by Trim-Away is acute and rapid, with half-lives of ~10-20 min. The major advantages of Trim-Away over other protein degradation methods are that it can be applied to any endogenous protein without prior modification; that it uses conventional antibodies that are widely available; and that it can be applied to a wide range of cell types, including nondividing primary human cells, for which other loss-of-function assays are challenging. In this protocol, we describe the detailed procedures for antibody preparation and delivery in mouse oocytes and cultured cells via microinjection and electroporation. In addition, we provide recommendations for antibody selection and validation, and for the generation of TRIM21-overexpressing cell lines for cases in which endogenous TRIM21 is limited. A typical Trim-Away experiment takes just a few hours.The research leading to these results received financial support from the Medical Research Council (MC_U105192711 and MC_U105181010), the Max Planck Society, the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 241548, European Research Council (ERC) Starting Grant no. 337415 and a Wellcome Trust Investigator Award

Human BRCA1-BARD1 ubiquitin ligase activity counters chromatin barriers to DNA resection

The opposing activities of 53BP1 and BRCA1 influence pathway choice of DNA double-strand break repair. How BRCA1 counters the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2~ubiquitin. We demonstrate that BRCA1-BARD1’s ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitylation by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1 deficient cells. We show BRCA1-BARD1 function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin, optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning and the need for SMARCAD1 in Olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus BRCA1- BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair

Turnover of BRCA1 Involves in Radiation-Induced Apoptosis

Background: Germ-line mutations of the breast cancer susceptibility gene-1 (BRCA1) increase the susceptibility to tumorigenesis. The function of BRCA1 is to regulate critical cellular processes, including cell cycle progression, genomic integrity, and apoptosis. Studies on the regulation of BRCA1 have focused intensely on transcription and phosphorylation mechanisms. Proteolytic regulation of BRCA1 in response to stress signaling remains largely unknown. The manuscript identified a novel mechanism by which BRCA1 is regulated by the ubiquitin-dependent degradation in response to ionization. Methodology/Principal Findings: Here, we report that severe ionization triggers rapid degradation of BRCA1, which in turn results in the activation of apoptosis. Ionization-induced BRCA1 turnover is mediated via an ubiquitin-proteasomal pathway. The stabilization of BRCA1 significantly delays the onset of ionization-induced apoptosis. We have mapped the essential region on BRCA1, which mediates its proteolysis in response to ionization. Moreover, we have demonstrated that BRCA1 protein is most sensitive to degradation when ionization occurs during G2/M and S phase. Conclusions/Significance: Our results suggest that ubiquitin-proteasome plays an important role in regulating BRCA1 during genotoxic stress. Proteolytic regulation of BRCA1 involves in ionization-induced apoptosis. © 2010 Liu et al

The ERCC6 Gene and Age-Related Macular Degeneration

Background: Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the developed countries and is caused by both environmental and genetic factors. A recent study (Tuo et al., PNAS) reported an association between AMD and a single nucleotide polymorphism (SNP) (rs3793784) in the ERCC6 (NM_000124) gene. The risk allele also increased ERCC6 expression. ERCC6 is involved in DNA repair and mutations in ERCC6 cause Cockayne syndrome (CS). Amongst others, photosensitivity and pigmentary retinopathy are hallmarks of CS. Methodology/Principal Findings: Separate and combined data from three large AMD case-control studies and a prospective population-based study (The Rotterdam Study) were used to analyse the genetic association between ERCC6 and AMD (2682 AMD cases and 3152 controls). We also measured ERCC6 mRNA levels in retinal pigment epithelium (RPE) cells of healthy and early AMD affected human donor eyes. Rs3793784 conferred a small increase in risk for late AMD in the Dutch population (The Rotterdam and AMRO-NL study), but this was not replicated in two non-European studies (AREDS, Columbia University). In addition, the AMRO-NL study revealed no significant association for 9 other variants spanning ERCC6. Finally, we determined that ERCC6 expression in the human RPE did not depend on rs3793784 genotype, but, interestingly, on AMD status: Early AMD-affected donor eyes had a 50% lower ERCC6 expression than healthy donor eyes (P = 0.018). Conclusions/Significance: Our meta analysis of four Caucasian cohorts does not replicate the reported association between SNPs in ERCC6 and AMD. Nevertheless, our findings on ERCC6 expression in the RPE suggest that ERCC6 may be functionally involved in AMD. Combining our data with those of the literature, we hypothesize that the AMD-related reduced transcriptional activity of ERCC6 may be caused by diverse, small and heterogeneous genetic and/or environmental determinants

An Abundant Evolutionarily Conserved CSB-PiggyBac Fusion Protein Expressed in Cockayne Syndrome

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein

Origin and Evolution of TRIM Proteins: New Insights from the Complete TRIM Repertoire of Zebrafish and Pufferfish

Tripartite motif proteins (TRIM) constitute a large family of proteins containing a RING-Bbox-Coiled Coil motif followed by different C-terminal domains. Involved in ubiquitination, TRIM proteins participate in many cellular processes including antiviral immunity. The TRIM family is ancient and has been greatly diversified in vertebrates and especially in fish. We analyzed the complete sets of trim genes of the large zebrafish genome and of the compact pufferfish genome. Both contain three large multigene subsets - adding the hsl5/trim35-like genes (hltr) to the ftr and the btr that we previously described - all containing a B30.2 domain that evolved under positive selection. These subsets are conserved among teleosts. By contrast, most human trim genes of the other classes have only one or two orthologues in fish. Loss or gain of C-terminal exons generated proteins with different domain organizations; either by the deletion of the ancestral domain or, remarkably, by the acquisition of a new C-terminal domain. Our survey of fish trim genes in fish identifies subsets with different evolutionary dynamics. trims encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they evolve fast, often under positive selection, and they duplicate to create multigenic families. We could identify new combinations of domains, which epitomize how new trim classes appear by domain insertion or exon shuffling. Notably, we found that a cyclophilin-A domain replaces the B30.2 domain of a zebrafish fintrim gene, as reported in the macaque and owl monkey antiretroviral TRIM5α. Finally, trim genes encoding RBCC-B30.2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogues, which suggests that such trim genes may have been part of the ancestral MHC

Antiviral TRIMs: friend or foe in autoimmune and autoinflammatory disease?

The concept that viral sensing systems, via their ability to drive pro-inflammatory cytokine and interferon production, contribute to the development of autoimmune and autoinflammatory disease is supported by a wide range of clinical and experimental observations. Recently, the tripartite motif-containing proteins (TRIMs) have emerged as having key roles in antiviral immunity — either as viral restriction factors or as regulators of pathways downstream of viral RNA and DNA sensors, and the inflammasome. Given their involvement in these pathways, we propose that TRIM proteins contribute to the development and pathology of autoimmune and autoinflammatory conditions, thus making them potential novel targets for therapeutic manipulation