Epigenetic Compound Screening Uncovers Small Molecules for Reactivation of Latent HIV-1.

During infection with the human immunodeficiency virus type 1 (HIV-1), latent reservoirs are established that circumvent full eradication of the virus by antiretroviral therapy (ART) and are the source for viral rebound after cessation of therapy. As these reservoirs are phenotypically indistinguishable from infected cells, current strategies aim to reactivate these reservoirs, followed by pharmaceutical and immunological destruction of the cells. Here, we employed a simple and convenient cell-based reporter system, which enables sample handling under biosafety level (BSL)-1 conditions, to screen for compounds that were able to reactivate latent HIV-1. The assay showed a high dynamic signal range and reproducibility with an average Z-factor of 0.77, classifying the system as robust. The assay was used for high-throughput screening (HTS) of an epigenetic compound library in combination with titration and cell-toxicity studies and revealed several potential new latency-reversing agents (LRAs). Further validation in well-known latency model systems verified earlier studies and identified two novel compounds with very high reactivation efficiencies and low toxicity. Both drugs, namely, N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) and 2',3'-difluoro-[1,1'-biphenyl]-4-carboxylic acid, 2-butylhydrazide (SR-4370), showed comparable performances to other already known LRAs, did not activate CD4+ T cells, and did not cause changes in the composition of peripheral blood mononuclear cells (PBMCs), as shown by flow cytometry analyses. Both compounds may represent effective new treatment possibilities for reversal of latency in HIV-1-infected individuals.

Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus.

ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a K(m) of 0.9 mm and k(cat) of 9 s(-1) at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli.

Strategies for investigating and optimizing the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

Mitochondrial ABC proteins in health and disease.

ABC transporters represent one of the largest families of membrane proteins that are found in all three phyla of life. Mitochondria comprise up to four ABC systems, ABCB7/ATM1, ABCB10/MDL1, ABCB8 and ABCB6. These half-transporters, which assemble into homodimeric complexes, are involved in a number of key cellular processes, e.g. biogenesis of cytosolic iron-sulfur clusters, heme biosynthesis, iron homeostasis, multidrug resistance, and protection against oxidative stress. Here, we summarize recent advances and emerging themes in our understanding of how these ABC systems in the inner and outer mitochondrial membrane fulfill their functions in important (patho) physiological processes, including neurodegenerative and hematological disorders.

SERINC5 Is an Unconventional HIV Restriction Factor That Is Upregulated during Myeloid Cell Differentiation.

Classical antiviral restriction factors promote cellular immunity by their ability to interfere with virus replication and induction of their expression by proinflammatory cytokines such as interferons. The serine incorporator proteins SERINC3 and SERINC5 potently reduce the infectivity of HIV-1 particles when overexpressed, and RNA interference or knockout approaches in T cells have indicated antiviral activity also of the endogenous proteins. Due to lack of reagents for detection of endogenous SERINC proteins, it is still unclear whether SERINC3/5 are expressed to functionally relevant levels in different primary target cells of HIV infection and how the expression levels of these innate immunity factors are regulated. In the current study, analysis of SERINC3/5 mRNA steady-state levels in primary lymphoid and monocyte-derived cells revealed selective induction of their expression upon differentiation of myeloid cells. Contrary to classical antiviral restriction factors, various antiviral α-interferon subtypes and proinflammatory interleukins had no effect on SERINC levels, which were also not dysregulated in CD4+ T cells and monocytes isolated from patients with chronic HIV-1 infection. Notably, HIV-1 particles produced by terminally differentiated monocyte-derived macrophages with high SERINC5 expression, but not by low-expressing monocytes, showed a Nef-dependent infectivity defect. Overall, these findings suggest endogenous expression of SERINC5 to antivirally active levels in macrophages. Our results classify SERINC5 as an unconventional HIV-1 restriction factor whose expression is specifically induced upon differentiation of cells towards the myeloid lineage.

Switching of the homooligomeric ATP-binding cassette transport complex MDL1 from post-translational mitochondrial import to endoplasmic reticulum insertion.

The ATP-binding cassette transporter MDL1 of Saccharomyces cerevisiae has been implicated in mitochondrial quality control, exporting degradation products of misassembled respiratory chain complexes. In the present study, we identified an unusually long leader sequence of 59 amino acids, which targets MDL1 to the inner mitochondrial membrane with its nucleotide-binding domain oriented to the matrix. By contrast, MDL1 lacking this leader sequence is directed into the endoplasmic reticulum membrane with the nucleotide-binding domain facing the cytosol. Remarkably, in both targeting routes, the ATP-binding cassette transporter maintains its intrinsic properties of membrane insertion and assembly, leading to homooligomeric complexes with similar activities in ATP hydrolysis. The physiological consequences of both targeting routes were elucidated in cells lacking the mitochondrial ATP-binding cassette transporter ATM1, which is essential for biogenesis of cytosolic iron-sulfur proteins. The mitochondrial MDL1 complex can complement ATM1 function, whereas the endoplasmic reticulum-targeted version, as well as MDL1 mutants deficient in ATP binding and hydrolysis, cannot overcome the Deltaatm1 growth phenotype.

Structural and functional fingerprint of the mitochondrial ATP-binding cassette transporter Mdl1 from Saccharomyces cerevisiae.

The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 microm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.