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Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.

Summary: Molecular chaperones such as heat-shock proteins (HSPs) help in protein folding. Their function in the cytosol has been well studied. Notably, chaperones are also present in the nucleus, a compartment where proteins enter after completing de novo folding in the cytosol, and this raises an important question about chaperone function in the nucleus. We performed a systematic analysis of the nuclear pool of heat-shock protein 90. Three orthogonal and independent analyses led us to the core functional interactome of HSP90. Computational and biochemical analyses identify host cell factor C1 (HCFC1) as a transcriptional regulator that depends on HSP90 for its stability. HSP90 was required to maintain the expression of HCFC1-targeted cell-cycle genes. The regulatory nexus between HSP90 and the HCFC1 module identified in this study sheds light on the relevance of chaperones in the transcription of cell-cycle genes. Our study also suggests a therapeutic avenue of combining chaperone and transcription inhibitors for cancer treatment. : What do chaperones do in the nucleus? Antonova et al. perform a comprehensive genetic and physical interactome analysis of nuclear HSP90 in human cells. HSP90 stabilizes the HCFC1 complex at chromatin, contributing to the expression of HCFC1-targeted cell-cycle genes. The simultaneous inhibition of HSP90 and transcription is synergistic in killing cancer cells. Keywords: chaperone, HSP90, HCFC1, chromatin, cancer, synergistic inhibition

Protein aggregation is associated with neurodegeneration and various other pathologies. How specific cellular environments modulate the aggregation of disease proteins is not well understood. Here, we investigated how the endoplasmic reticulum (ER) quality control system handles beta-sheet proteins that were designed de novo to form amyloid-like fibrils. While these proteins undergo toxic aggregation in the cytosol, we find that targeting them to the ER (ER-beta) strongly reduces their toxicity. ER-beta is retained within the ER in a soluble, polymeric state, despite reaching very high concentrations exceeding those of ER-resident molecular chaperones. ER-beta is not removed by ER-associated degradation (ERAD) but interferes with ERAD of other proteins. These findings demonstrate a remarkable capacity of the ER to prevent the formation of insoluble beta-aggregates and the secretion of potentially toxic protein species. Our results also suggest a generic mechanism by which proteins with exposed b-sheet structure in the ER interfere with proteostasis.

SAMHD1 is a GTP-activated nonspecific dNTP triphosphohydrolase that depletes dNTP pools in resting CD4+ T cells and macrophages and effectively restricts infection by HIV-1. We have designed a nonsubstrate dUTP analogue with a methylene bridge connecting the α phosphate and 5′ carbon that potently inhibits SAMHD1. Although pppCH2dU shows apparent competitive inhibition, it acts by a surprising allosteric mechanism that destabilizes active enzyme tetramer.

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001, eLife digest Human immunodeficiency virus type 1 (HIV-1) infects and kills immune cells known as CD4+ T cells, leading to the disease AIDS. Current drug treatments enable HIV-1 infected patients to live relatively long and healthy lives. However, no cure for HIV-1 exists because the virus lives indefinitely in a resting state within the genetic material – or genome – of the infected cell, where it is not susceptible to drug treatments. Most HIV-1 research focuses on T cells, but another type of immune cell – the macrophage – may also harbor resting HIV-1 in its genome. Compared to other cells, macrophages are unusual because they produce large amounts of a molecule called deoxyuridine triphosphate (dUTP). Most cells, including T cells, keep dUTP levels very low because it closely resembles molecules that are used to make DNA and so it can be accidentally incorporated into the cell’s DNA. When this happens, the cell removes the dUTP from the DNA using enzymes in a process called uracil base excision repair (UBER). To hide inside the cell’s genome, HIV-1 needs to produce a DNA copy of its own genome, but it was not known what happens when HIV-1 tries to do this within a macrophage that contains high levels of dUTP and UBER enzymes. Here, Hansen et al. reveal that about 90% of macrophages have exceptionally high levels of dUTP and are poorly infected by HIV-1. The high levels of dUTP result in the virus incorporating dUTP into its DNA, which is then attacked and fragmented by UBER enzymes. However, about one in a hundred viral DNA molecules do manage to successfully integrate into the genome of the macrophage. This viral DNA later gives rise to new virus particles through an error-prone process that, by introducing new mutations into the virus genome, may help HIV-1 to evolve and persist. Further experiments examined cells that give rise to macrophages from infected patients who had been on anti-HIV drug therapy for several years. Hansen et al. found that there was lots of dUTP in the DNA sequences of HIV-1 viruses found in these “precursor” cells. These precursor cells only live for several days before being eliminated, so the presence of viruses containing dUTP suggests these cells were infected recently. A future challenge will be to identify new anti-HIV drugs that specifically target macrophages and to understand the role of error-prone production of new viral genomes. DOI: http://dx.doi.org/10.7554/eLife.18447.002

We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein-protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors.

The HIV-1 restriction factor sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a tetrameric protein that catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and tripolyphosphate, which effectively depletes the dNTP substrates of HIV reverse transcriptase. Here, we establish that SAMHD1 is activated by GTP binding to guanine-specific activator sites (A1) as well as coactivation by substrate dNTP binding to a distinct set of nonspecific activator sites (A2). Combined activation by GTP and dNTPs results in a long-lived tetrameric form of SAMHD1 that persists for hours, even after activating nucleotides are withdrawn from the solution. These results reveal an ordered model for assembly of SAMHD1 tetramer from its inactive monomer and dimer forms, where GTP binding to the A1 sites generates dimer and dNTP binding to the A2 and catalytic sites generates active tetramer. Thus, cellular regulation of active SAMHD1 is not determined by GTP alone but instead, the levels of all dNTPs and the generation of a persistent tetramer that is not in equilibrium with free activators. The significance of the long-lived activated state is that SAMHD1 can remain active long after dNTP pools have been reduced to a level that would lead to inactivation. This property would be important in resting CD4(+) T cells, where dNTP pools are reduced to nanomolar levels to restrict infection by HIV-1.

Page 4702. In Figure 2, the chemical structure of compound 19 (DG-051) was incorrectly drawn as the (R)-enantiomer. Compound 19 is the (S)-enantiomer and is properly shown in the three-dimensional crystal structure image at the right-hand side in the corrected figure below. Figure 2 Panels showing ligand binding to LTA4H for compounds described in the manuscript. Enzyme assay IC50 values are in μM, human whole blood cell assay IC50 values are in nM when reported, and ligand efficiency (LE) values are in kcal/(mol·heavy atom). Compound structures are displayed as yellow stick structures, and LTA4H is displayed in gray. Green mesh corresponds to the Fo − Fc (difference) electron density at the 3.0σ level of the crystal structure with the compound omitted from the model. Polar contacts with LTA4H and/or bound water molecules are shown as red dashed lines. PDB IDs for each structure are indicated. On page 4704, right hand column, first paragraph, line 19 and second paragraph line 22, (R)-prolinol should be changed to (S)-prolinol. Supporting Information Page S16. In Table 2, the chemical structure of compound 19 (DG-051) was incorrectly drawn as the (R)-enantiomer. The correct (S)-enantiomer has been included in the revised version of Supplemental Table 2 below. Supplemental Table 2. Comparative IC50 data for peptidase, hydrolase, and HWB assays. View it in a separate window Supplemental Table 2. Comparative IC50data for peptidase, hydrolase, and HWB assays. For all compound structures described in this work, inhibition assays were carried out using at least two independent methods, either the hydrolase assay, peptidase assay and/or a hydrolase assay in human whole blood. See Materials and Methods section for details. All results are given as IC50 values in μM, with the number of replicates and the standard error of the mean indicated where applicable.