SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage ...virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.
SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a ...level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.
Transcription of endogenous retroviral elements are tightly regulated during development by members of the KRAB‐containing zinc finger proteins (KRAB‐ZFPs) and the co‐repressor Trim28 (also known as ...Kap‐1 or Tif1β). KRAB‐ZFPs form the largest family of transcription regulators in mammals and initiate transcriptional silencing by tethering Trim28 to a target locus. Subsequently, Trim28 recruits chromatin modifying effectors resulting in the formation of heterochromatin. In the present study, we identify surface exposed residues on the central six turns of the Trim28 coiled‐coil region forming the binding interface for the KRAB domain. Using AlphaFold2 (AF2) we provide high confidence models of the interface between Trim28 and the KRAB domain and identified leucine 301 on each chain of the Trim28 monomer to act as a pin extending into a hydrophobic pocket on the KRAB domain surface. Site directed mutations in the Trim28‐KRAB binding interface abolished binding to the KRAB domain. Our work provides a detailed understanding of the specific interactions between the KRAB domain and the Trim28 coiled‐coil and how this interaction may be regulated during silencing events.
Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as ...single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish cyp20a1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1-/- mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.
On the occurrence of cytochrome P450 in viruses Lamb, David C.; Follmer, Alec H.; Goldstone, Jared V. ...
Proceedings of the National Academy of Sciences - PNAS,
06/2019, Volume:
116, Issue:
25
Journal Article
Peer reviewed
Open access
Genes encoding cytochrome P450 (CYP; P450) enzymes occur widely in the Archaea, Bacteria, and Eukarya, where they play important roles in metabolism of endogenous regulatory molecules and exogenous ...chemicals. We now report that genes for multiple and unique P450s occur commonly in giant viruses in the Mimiviridae, Pandoraviridae, and other families in the proposed order Megavirales. P450 genes were also identified in a herpesvirus (Ranid herpesvirus 3) and a phage (Mycobacterium phage Adler). The Adler phage P450 was classified as CYP102L1, and the crystal structure of the open formwas solved at 2.5 Å. Genes encoding known redox partners for P450s (cytochrome P450 reductase, ferredoxin and ferredoxin reductase, and flavodoxin and flavodoxin reductase) were not found in any viral genome so far described, implying that host redox partners may drive viral P450 activities. Giant virus P450 proteins share no more than 25% identity with the P450 gene products we identified in Acanthamoeba castellanii, an amoeba host for many giant viruses. Thus, the origin of the unique P450 genes in giant viruses remains unknown. If giant virus P450 genes were acquired from a host, we suggest it could have been from an as yet unknown and possibly ancient host. These studies expand the horizon in the evolution and diversity of the enormously important P450 superfamily. Determining the origin and function of P450s in giant viruses may help to discern the origin of the giant viruses themselves.
Peroxiredoxins are antioxidant proteins primarily responsible for detoxification of hydroperoxides in cells. On exposure to various cellular stresses, peroxiredoxins can acquire chaperone activity, ...manifested as quaternary reorganization into a high molecular weight (HMW) form. Acidification, for example, causes dodecameric rings of human peroxiredoxin 3 (HsPrx3) to stack into long helical filaments. In this work, a 4.1-Å resolution structure of low-pH-instigated helical filaments was elucidated, showing a locally unfolded active site and partially folded C terminus. A 2.8-Å crystal structure of HsPrx3 was determined at pH 8.5 under reducing conditions, wherein dodecameric rings are arranged as a short stack, with symmetry similar to low-pH filaments. In contrast to previous observations, the crystal structure displays both a fully folded active site and ordered C terminus, suggesting that the HsPrx3 HMW form maintains catalytic activity. We propose a new role for the HMW form as a self-chaperoning assembly maintaining HsPrx3 function under stress.
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•Human peroxiredoxin 3 structures determined using cryo-EM and X-ray crystallography•Crystal structure at pH 8.5 displays helical stacking similar to pH 4.0 EM assembly•Active sites of HsPrx3 stacks are locally unfolded (pH 4.0) and fully folded (pH 8.5)•HMW form retains catalytic state, suggesting self-associating chaperone function
Yewdall et al. use cryo-EM and X-ray crystallography to reveal that human peroxiredoxin 3 forms HMW helical filaments at pH 4.0 and pH 8.5. The active-site conformations were locally unfolded (pH 4.0) and fully folded (pH 8.5), suggesting that the HsPrx3 HMW form maintains catalytic activity and could function as a putative self-assembling chaperone.
Trichomonas vaginalis is a human protozoan parasite that causes trichomoniasis, a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a shift to a dysbiotic vaginal microbiome ...that is depleted of lactobacilli. Studies on co-cultures have shown that vaginal bacteria in eubiosis (e.g. Lactobacillus gasseri) have antagonistic effects on T. vaginalis pathogenesis, suggesting that the parasite might benefit from shaping the microbiome to dysbiosis (e.g. Gardnerella vaginalis among other anaerobes). We have recently shown that T. vaginalis has acquired NlpC/P60 genes from bacteria, expanding them to a repertoire of nine TvNlpC genes in two distinct clans, and that TvNlpCs of clan A are active against bacterial peptidoglycan. Here, we expand this characterization to TvNlpCs of clan B. In this study, we show that the clan organisation of NlpC/P60 genes is a feature of other species of Trichomonas, and that Histomonas meleagridis has sequences related to one clan. We characterized the 3D structure of TvNlpC_B3 alone and with the inhibitor E64 bound, probing the active site of these enzymes for the first time. Lastly, we demonstrated that TvNlpC_B3 and TvNlpC_B5 have complementary activities with the previously described TvNlpCs of clan A and that exogenous expression of these enzymes empower this mucosal parasite to take over populations of vaginal lactobacilli in mixed cultures. TvNlpC_B3 helps control populations of L. gasseri, but not of G. vaginalis, which action is partially inhibited by E64. This study is one of the first to show how enzymes produced by a mucosal protozoan parasite may contribute to a shift on the status of a microbiome, helping explain the link between trichomoniasis and vaginal dysbiosis. Further understanding of this process might have significant implications for treatments in the future.
While most Rh–N-heterocyclic carbene (NHC) complexes currently investigated in anticancer research contain a Rh(III) metal center, an increasing amount of research is focusing on the cytotoxic ...activity and mode of action of square-planar RhCl(COD)(NHC) (where COD = 1,5-cyclooctadiene) which contains a Rh(I) center. The enzyme thioredoxin reductase (TrxR) and the protein albumin have been proposed as potential targets, but the molecular processes taking place upon protein interaction remain elusive. Herein, we report the preparation of peptide-conjugated and its nonconjugated parent RhCl(COD)(NHC) complexes, an in-depth investigation of both their stability in solution, and a crystallographic study of protein interaction. The organorhodium compounds showed a rapid loss of the COD ligand and slow loss of the NHC ligand in aqueous solution. These ligand exchange reactions were reflected in studies on the interaction with hen egg white lysozyme (HEWL) as a model protein in single-crystal X-ray crystallographic investigations. Upon treatment of HEWL with an amino acid functionalized RhCl(COD)(NHC) complex, two distinct rhodium adducts were found initially after 7 d of incubation at His15 and after 4 weeks also at Lys33. In both cases, the COD and chlorido ligands had been substituted with aqua and/or hydroxido ligands. While the histidine (His) adduct also indicated a loss of the NHC ligand, the lysine (Lys) adduct retained the NHC core derived from the amino acid l-histidine. In either case, an octahedral coordination environment of the metal center indicates oxidation to Rh(III). This investigation gives the first insight on the interaction of Rh(I)(NHC) complexes and proteins at the molecular level.
Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact ...mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA‐C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as Ru(cym), and Ru(cym) or Ru(cym)(PTA) adducts, respectively. Myoglobin bound up to four Ru(cym)(PTA) moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision‐induced unfolding (CIU) studies via ion‐mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein–RAPTA‐C adducts showed binding of Ru(cym) to Met1 caused a significant compaction of ubiquitin, likely from N‐terminal S−Ru−N chelation, while binding of Ru(cym)(PTA) to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds.
Mass spectrometric studies showed that the anticancer agent RAPTA‐C binds most effectively to ubiquitin and myoglobin over cytochrome c and lysozyme via coordination to His and/or Met residues. While coordination of Ru(cym) to ubiquitin at Met1 caused a large compaction of the native‐like protein, Ru(cym)(PTA) adducts had negligible effects, as also observed for myoglobin.
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