Publications by authors named "Maria DeLucia"

22 Publications

  • Page 1 of 1

HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7.

Virol J 2021 Mar 1;18(1):48. Epub 2021 Mar 1.

Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, RM 1055, 3501 Fifth Ave., Pittsburgh, PA, 15260, USA.

Background: Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1.

Methods: HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein-protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting.

Results: We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, CONCLUSIONS: Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.
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http://dx.doi.org/10.1186/s12985-021-01514-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923639PMC
March 2021

Complete H, C, N resonance assignments and secondary structure of the Vpr binding region of hHR23A (residues 223-363).

Biomol NMR Assign 2020 04 28;14(1):13-17. Epub 2019 Aug 28.

Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Ave., Pittsburgh, PA, 15261, USA.

Comprehensive resonance assignments and delineation of the secondary structure elements of the C-terminal Vpr-binding region of hHR23A, residues 223-363, were achieved by triple-resonance NMR experiments on uniformly C,N-labeled protein. Assignments are 100% and > 95% complete for backbone and side-chain resonances, respectively. This data constitutes important complementary information for our ongoing structure determination of the Vpr-hHR23A(223-363) complex. At high concentrations, severe line-broadening was observed for several residues in the H-N HSQC spectrum, most likely resulting from inter-molecular interactions.
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http://dx.doi.org/10.1007/s12104-019-09913-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047585PMC
April 2020

Surface-Binding to Cardiolipin Nanodomains Triggers Cytochrome c Pro-apoptotic Peroxidase Activity via Localized Dynamics.

Structure 2019 05 14;27(5):806-815.e4. Epub 2019 Mar 14.

Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA; Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands. Electronic address:

The peroxidation of cardiolipins by reactive oxygen species, which is regulated and enhanced by cytochrome c (cyt c), is a critical signaling event in mitochondrial apoptosis. We probe the molecular underpinnings of this mitochondrial death signal through structural and functional studies of horse heart cyt c binding to mixed-lipid membranes containing cardiolipin with mono- and polyunsaturated acyl chains. Lipidomics reveal the selective oxidation of polyunsaturated fatty acid (PUFA) cardiolipin (CL), while multidimensional solid-state NMR probes the structure and dynamics of the membrane and the peripherally bound protein. The hydrophilic milieu at the membrane interface stabilizes a native-like fold, but also leads to localized flexibility at the membrane-interacting protein face. PUFA CL acts as both a preferred substrate and a dynamic regulator by affecting the dynamics of the cyt c N70-I85 Ω loop, which covers the heme cavity.
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http://dx.doi.org/10.1016/j.str.2019.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615723PMC
May 2019

HIV-1 Vpr Reprograms CLR4 E3 Ubiquitin Ligase to Antagonize Exonuclease 1-Mediated Restriction of HIV-1 Infection.

mBio 2018 10 23;9(5). Epub 2018 Oct 23.

Department of Molecular Biology and Microbiology, Case Western Reserve School of Medicine, Cleveland, Ohio, USA

Viral accessory proteins hijack host cell E3 ubiquitin ligases to antagonize innate/intrinsic defenses and thereby provide a more permissive environment for virus replication. Human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr reprograms CRL4 E3 to antagonize select postreplication DNA repair enzymes, but the significance and role of these Vpr interactions are poorly understood. To gain additional insights, we performed a focused screen for substrates of CRL4 E3 reprogrammed by HIV-1 Vpr among known postreplication DNA repair proteins and identified exonuclease 1 (Exo1) as a novel direct HIV-1 Vpr target. We show that HIV-1 Vpr recruits Exo1 to the CRL4 E3 complex for ubiquitination and subsequent proteasome-dependent degradation and that Exo1 levels are depleted in HIV-1-infected cells in a Vpr-dependent manner. We also show that Exo1 inhibits HIV-1 replication in T cells. Notably, the antagonism of Exo1 is a conserved function of main group HIV-1 and its ancestor Vpr proteins in the simian immunodeficiency virus from chimpanzee (SIVcpz) lineage, further underscoring the relevance of our findings. Overall, our studies (i) reveal that HIV-1 Vpr extensively remodels the cellular postreplication DNA repair machinery by impinging on multiple repair pathways, (ii) support a model in which Vpr promotes HIV-1 replication by antagonizing select DNA repair enzymes, and (iii) highlight the importance of a new class of restrictions placed on HIV-1 replication in T cells by the cellular DNA repair machinery. HIV-1 polymerase reverse transcribes the viral RNA genome into imperfectly double-stranded proviral DNA, containing gaps and flaps, for integration into the host cell chromosome. HIV-1 reverse transcripts share characteristics with cellular DNA replication intermediates and are thought to be converted into fully double-stranded DNA by cellular postreplication DNA repair enzymes. Therefore, the finding that the HIV-1 accessory protein Vpr antagonizes select postreplication DNA repair enzymes that can process HIV-1 reverse transcripts has been surprising. Here, we show that one such Vpr-antagonized enzyme, exonuclease 1, inhibits HIV-1 replication in T cells. We identify exonuclease 1 as a member of a new class of HIV-1 restriction factors in T cells and propose that certain modes of DNA "repair" inhibit HIV-1 infection.
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http://dx.doi.org/10.1128/mBio.01732-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199497PMC
October 2018

HIV-1 Vpr protein directly loads helicase-like transcription factor (HLTF) onto the CRL4-DCAF1 E3 ubiquitin ligase.

J Biol Chem 2017 12 27;292(51):21117-21127. Epub 2017 Oct 27.

From the Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260 and

The viral protein R (Vpr) is an accessory virulence factor of HIV-1 that facilitates infection in immune cells. Cellular functions of Vpr are tied to its interaction with DCAF1, a substrate receptor component of the CRL4 E3 ubiquitin ligase. Recent proteomic approaches suggested that Vpr degrades helicase-like transcription factor (HLTF) DNA helicase in a proteasome-dependent manner by redirecting the CRL4-DCAF1 E3 ligase. However, the precise molecular mechanism of Vpr-dependent HLTF depletion is not known. Here, using reconstitution assays, we show that Vpr mediates polyubiquitination of HLTF, by directly loading it onto the C-terminal WD40 domain of DCAF1 in complex with the CRL4 E3 ubiquitin ligase. Mutational analyses suggest that Vpr interacts with DNA-binding residues in the N-terminal HIRAN domain of HLTF in a manner similar to the recruitment of another target, uracil DNA glycosylase (UNG2), to the CRL4-DCAF1 E3 by Vpr. Strikingly, Vpr also engages a second, adjacent region, which connects the HIRAN and ATPase/helicase domains. Thus, our findings reveal that Vpr utilizes common as well as distinctive interfaces to recruit multiple postreplication DNA repair proteins to the CRL4-DCAF1 E3 ligase for ubiquitin-dependent proteasomal degradation.
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http://dx.doi.org/10.1074/jbc.M117.798801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743084PMC
December 2017

The DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction.

Nat Struct Mol Biol 2016 Oct 29;23(10):933-940. Epub 2016 Aug 29.

Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

The HIV-1 accessory protein Vpr is required for efficient viral infection of macrophages and promotion of viral replication in T cells. Vpr's biological activities are closely linked to the interaction with human DCAF1, a cellular substrate receptor of the Cullin4-RING E3 ubiquitin ligase (CRL4) of the host ubiquitin-proteasome-mediated protein degradation pathway. The molecular details of how Vpr usurps the protein degradation pathway have not been delineated. Here we present the crystal structure of the DDB1-DCAF1-HIV-1-Vpr-uracil-DNA glycosylase (UNG2) complex. The structure reveals how Vpr engages with DCAF1, creating a binding interface for UNG2 recruitment in a manner distinct from the recruitment of SAMHD1 by Vpx proteins. Vpr and Vpx use similar N-terminal and helical regions to bind the substrate receptor, whereas different regions target the specific cellular substrates. Furthermore, Vpr uses molecular mimicry of DNA by a variable loop for specific recruitment of the UNG2 substrate.
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http://dx.doi.org/10.1038/nsmb.3284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385928PMC
October 2016

SLX4-SLX1 Protein-independent Down-regulation of MUS81-EME1 Protein by HIV-1 Viral Protein R (Vpr).

J Biol Chem 2016 08 27;291(33):16936-16947. Epub 2016 Jun 27.

From the Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260

Evolutionarily conserved structure-selective endonuclease MUS81 forms a complex with EME1 and further associates with another endonuclease SLX4-SLX1 to form a four-subunit complex of MUS81-EME1-SLX4-SLX1, coordinating distinctive biochemical activities of both endonucleases in DNA repair. Viral protein R (Vpr), a highly conserved accessory protein in primate lentiviruses, was previously reported to bind SLX4 to mediate down-regulation of MUS81. However, the detailed mechanism underlying MUS81 down-regulation is unclear. Here, we report that HIV-1 Vpr down-regulates both MUS81 and its cofactor EME1 by hijacking the host CRL4-DCAF1 E3 ubiquitin ligase. Multiple Vpr variants, from HIV-1 and SIV, down-regulate both MUS81 and EME1. Furthermore, a C-terminally truncated Vpr mutant and point mutants R80A and Q65R, all of which lack G2 arrest activity, are able to down-regulate MUS81-EME1, suggesting that Vpr-induced G2 arrest is not correlated with MUS81-EME1 down-regulation. We also show that neither the interaction of MUS81-EME1 with Vpr nor their down-regulation is dependent on SLX4-SLX1. Together, these data provide new insight on a conserved function of Vpr in a host endonuclease down-regulation.
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http://dx.doi.org/10.1074/jbc.M116.721183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016100PMC
August 2016

Structural Changes and Proapoptotic Peroxidase Activity of Cardiolipin-Bound Mitochondrial Cytochrome c.

Biophys J 2015 Nov;109(9):1873-84

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Electronic address:

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.
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http://dx.doi.org/10.1016/j.bpj.2015.09.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643272PMC
November 2015

Structural Basis of Clade-specific Engagement of SAMHD1 (Sterile α Motif and Histidine/Aspartate-containing Protein 1) Restriction Factors by Lentiviral Viral Protein X (Vpx) Virulence Factors.

J Biol Chem 2015 Jul 4;290(29):17935-17945. Epub 2015 Jun 4.

Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260. Electronic address:

Sterile α motif (SAM) and histidine/aspartate (HD)-containing protein 1 (SAMHD1) restricts human/simian immunodeficiency virus infection in certain cell types and is counteracted by the virulence factor Vpx. Current evidence indicates that Vpx recruits SAMHD1 to the Cullin4-Ring Finger E3 ubiquitin ligase (CRL4) by facilitating an interaction between SAMHD1 and the substrate receptor DDB1- and Cullin4-associated factor 1 (DCAF1), thereby targeting SAMHD1 for proteasome-dependent down-regulation. Host-pathogen co-evolution and positive selection at the interfaces of host-pathogen complexes are associated with sequence divergence and varying functional consequences. Two alternative interaction interfaces are used by SAMHD1 and Vpx: the SAMHD1 N-terminal tail and the adjacent SAM domain or the C-terminal tail proceeding the HD domain are targeted by different Vpx variants in a unique fashion. In contrast, the C-terminal WD40 domain of DCAF1 interfaces similarly with the two above complexes. Comprehensive biochemical and structural biology approaches permitted us to delineate details of clade-specific recognition of SAMHD1 by lentiviral Vpx proteins. We show that not only the SAM domain but also the N-terminal tail engages in the DCAF1-Vpx interaction. Furthermore, we show that changing the single Ser-52 in human SAMHD1 to Phe, the residue found in SAMHD1 of Red-capped monkey and Mandrill, allows it to be recognized by Vpx proteins of simian viruses infecting those primate species, which normally does not target wild type human SAMHD1 for degradation.
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http://dx.doi.org/10.1074/jbc.M115.665513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505041PMC
July 2015

CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain.

J Biol Chem 2015 May 6;290(21):13279-92. Epub 2015 Apr 6.

From the Department of Molecular Biology and Microbiology, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260,

SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication.
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http://dx.doi.org/10.1074/jbc.M115.646588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505580PMC
May 2015

Structural basis of allosteric activation of sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) by nucleoside triphosphates.

J Biol Chem 2014 Nov 6;289(47):32617-27. Epub 2014 Oct 6.

From the Department of Structural Biology and Pittsburgh Center for HIV-Host Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260

Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) plays a critical role in inhibiting HIV infection, curtailing the pool of dNTPs available for reverse transcription of the viral genome. Recent structural data suggested a compelling mechanism for the regulation of SAMHD1 enzymatic activity and revealed dGTP-induced association of two inactive dimers into an active tetrameric enzyme. Here, we present the crystal structures of SAMHD1 catalytic core (residues 113-626) tetramers, complexed with mixtures of nucleotides, including dGTP/dATP, dGTP/dCTP, dGTP/dTTP, and dGTP/dUTP. The combined structural and biochemical data provide insight into dNTP promiscuity at the secondary allosteric site and how enzymatic activity is modulated. In addition, we present biochemical analyses of GTP-induced SAMHD1 full-length tetramerization and the structure of SAMHD1 catalytic core tetramer in complex with GTP/dATP, revealing the structural basis of GTP-mediated SAMHD1 activation. Altogether, the data presented here advance our understanding of SAMHD1 function during cellular homeostasis.
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http://dx.doi.org/10.1074/jbc.M114.591958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239615PMC
November 2014

Mechanisms of allosteric activation and inhibition of the deoxyribonucleoside triphosphate triphosphohydrolase from Enterococcus faecalis.

J Biol Chem 2014 Jan 12;289(5):2815-24. Epub 2013 Dec 12.

From the Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611 and.

EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.
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http://dx.doi.org/10.1074/jbc.M113.524207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908413PMC
January 2014

Binding of HIV-1 Vpr protein to the human homolog of the yeast DNA repair protein RAD23 (hHR23A) requires its xeroderma pigmentosum complementation group C binding (XPCB) domain as well as the ubiquitin-associated 2 (UBA2) domain.

J Biol Chem 2014 Jan 8;289(5):2577-88. Epub 2013 Dec 8.

From the Department of Structural Biology and Pittsburgh Center for HIV-Host Protein Interactions, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261.

The human homolog of the yeast DNA repair protein RAD23, hHR23A, has been found previously to interact with the human immunodeficiency virus, type 1 accessory protein Vpr. hHR23A is a modular protein containing an N-terminal ubiquitin-like (UBL) domain and two ubiquitin-associated domains (UBA1 and UBA2) separated by a xeroderma pigmentosum complementation group C binding (XPCB) domain. All domains are connected by flexible linkers. hHR23A binds ubiquitinated proteins and acts as a shuttling factor to the proteasome. Here, we show that hHR23A utilizes both the UBA2 and XPCB domains to form a stable complex with Vpr, linking Vpr directly to cellular DNA repair pathways and their probable exploitation by the virus. Detailed structural mapping of the Vpr contacts on hHR23A, by NMR, revealed substantial contact surfaces on the UBA2 and XPCB domains. In addition, Vpr binding disrupts an intramolecular UBL-UBA2 interaction. We also show that Lys-48-linked di-ubiquitin, when binding to UBA1, does not release the bound Vpr from the hHR23A-Vpr complex. Instead, a ternary hHR23A·Vpr·di-Ub(K48) complex is formed, indicating that Vpr does not necessarily abolish hHR23A-mediated shuttling to the proteasome.
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http://dx.doi.org/10.1074/jbc.M113.534453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908392PMC
January 2014

Mechanism of allosteric activation of SAMHD1 by dGTP.

Nat Struct Mol Biol 2013 Nov 20;20(11):1304-9. Epub 2013 Oct 20.

1] Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA. [2].

SAMHD1, a dNTP triphosphohydrolase (dNTPase), has a key role in human innate immunity. It inhibits infection of blood cells by retroviruses, including HIV, and prevents the development of the autoinflammatory Aicardi-Goutières syndrome (AGS). The inactive apo-SAMHD1 interconverts between monomers and dimers, and in the presence of dGTP the protein assembles into catalytically active tetramers. Here, we present the crystal structure of the human tetrameric SAMHD1-dGTP complex. The structure reveals an elegant allosteric mechanism of activation through dGTP-induced tetramerization of two inactive dimers. Binding of dGTP to four allosteric sites promotes tetramerization and induces a conformational change in the substrate-binding pocket to yield the catalytically active enzyme. Structure-based biochemical and cell-based biological assays confirmed the proposed mechanism. The SAMHD1 tetramer structure provides the basis for a mechanistic understanding of its function in HIV restriction and the pathogenesis of AGS.
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http://dx.doi.org/10.1038/nsmb.2692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3833828PMC
November 2013

HIV-2 and SIVmac accessory virulence factor Vpx down-regulates SAMHD1 enzyme catalysis prior to proteasome-dependent degradation.

J Biol Chem 2013 Jun 15;288(26):19116-26. Epub 2013 May 15.

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.

SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and simian immunodeficiency virus (SIV) counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for polyubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known. We applied surface plasmon resonance analysis to assess the requirements for and kinetics of binding between various primate SAMHD1 proteins and Vpx proteins from SIV or HIV-2 strains. Our data indicate that Vpx proteins, bound to DCAF1, interface with the C terminus of primate SAMHD1 proteins with nanomolar affinity, manifested by rapid association and slow dissociation. Further, we provide evidence that Vpx binding to SAMHD1 inhibits its catalytic activity and induces disassembly of a dGTP-dependent oligomer. Our studies reveal a previously unrecognized biochemical mechanism of Vpx-mediated SAMHD1 inhibition: direct down-modulation of its catalytic activity, mediated by the same binding event that leads to SAMHD1 recruitment to the E3 ubiquitin ligase for proteasome-dependent degradation.
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http://dx.doi.org/10.1074/jbc.M113.469007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696684PMC
June 2013

Tetramerization of SAMHD1 is required for biological activity and inhibition of HIV infection.

J Biol Chem 2013 Apr 20;288(15):10406-17. Epub 2013 Feb 20.

Department of Molecular Biology and Microbiology, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA.

SAMHD1 is a dGTP-activated dNTPase that has been implicated as a modulator of the innate immune response. In monocytes and their differentiated derivatives, as well as in quiescent cells, SAMHD1 strongly inhibits HIV-1 infection and, to a lesser extent, HIV-2 and simian immunodeficiency virus (SIV) because of their virion-associated virulence factor Vpx, which directs SAMHD1 for proteasomal degradation. Here, we used a combination of biochemical and virologic approaches to gain insights into the functional organization of human SAMHD1. We found that the catalytically active recombinant dNTPase is a dGTP-induced tetramer. Chemical cross-linking studies revealed SAMHD1 tetramers in human monocytic cells, in which it strongly restricts HIV-1 infection. The propensity of SAMHD1 to maintain the tetrameric state in vitro is regulated by its C terminus, located outside of the catalytic domain. Accordingly, we show that the C terminus is required for the full ability of SAMHD1 to deplete dNTP pools and to inhibit HIV-1 infection in U937 monocytes. Interestingly, the human SAMHD1 C terminus contains a docking site for HIV-2/SIVmac Vpx and is known to have evolved under positive selection. This evidence indicates that Vpx targets a functionally important element in SAMHD1. Together, our findings imply that SAMHD1 tetramers are the biologically active form of this dNTPase and provide new insights into the functional organization of SAMHD1.
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http://dx.doi.org/10.1074/jbc.M112.443796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624423PMC
April 2013

Protease cleavage leads to formation of mature trimer interface in HIV-1 capsid.

PLoS Pathog 2012 23;8(8):e1002886. Epub 2012 Aug 23.

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.
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http://dx.doi.org/10.1371/journal.ppat.1002886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426514PMC
December 2012

HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.

J Biol Chem 2012 Apr 23;287(15):12550-8. Epub 2012 Feb 23.

Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.

The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4(DCAF1) E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4(DCAF1-Vpx) E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4(DCAF1) and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.
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http://dx.doi.org/10.1074/jbc.M112.340711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321004PMC
April 2012

A randomized controlled study of effects of dietary magnesium oxide supplementation on bone mineral content in healthy girls.

J Clin Endocrinol Metab 2006 Dec 3;91(12):4866-72. Epub 2006 Oct 3.

Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8064, USA.

Context: The role of magnesium (Mg) as a determinant of bone mass has not been extensively explored. Limited studies suggest that dietary Mg intake and bone mineral density are correlated in adults, but no data from interventional studies in children and adolescents are available.

Objective: We sought to determine whether Mg supplementation in periadolescent girls enhances accrual of bone mass.

Design: We carried out a prospective, placebo-controlled, randomized, one-year double-blind trial of Mg supplementation.

Setting: The study was conducted in the Clinical Research Centers at Yale University School of Medicine.

Patients Or Other Participants: Healthy 8- to 14-yr-old Caucasian girls were recruited from community pediatricians' offices. Dietary diaries from over 120 volunteers were analyzed, and those with dietary Mg intake of less than 220 mg/d were invited to participate in the intervention.

Intervention: Magnesium (300 mg elemental Mg per day in two divided doses) or placebo was given orally for 12 months.

Main Outcome Measure: The primary outcome measure was interval change in bone mineral content (BMC) of the total hip, femoral neck, Ward's area, and lumbar spine (L1-L4) after 12 months of Mg supplementation.

Results: Significantly increased accrual (P = 0.05) in integrated hip BMC occurred in the Mg-supplemented vs. placebo group. Trends for a positive Mg effect were evident in the pre- and early puberty and in mid-late puberty. Lumbar spinal BMC accrual was slightly (but not significantly) greater in the Mg-treated group. Compliance was excellent; 73% of capsules were ingested as inferred by pill counts. Serum mineral levels, calciotropic hormones, and bone markers were similar between groups.

Conclusions: Oral Mg oxide capsules are safe and well tolerated. A positive effect of Mg supplementation on integrated hip BMC was evident in this small cohort.
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http://dx.doi.org/10.1210/jc.2006-1391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995550PMC
December 2006

Nutritional rickets with normal circulating 25-hydroxyvitamin D: a call for reexamining the role of dietary calcium intake in North American infants.

J Clin Endocrinol Metab 2003 Aug;88(8):3539-45

Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8064, USA.

The incidence of nutritional rickets appears to be increasing in North American infants and toddlers; it is widely assumed that this is due to vitamin D deficiency. Thus, records of 43 children with nutritional rickets from greater New Haven, Connecticut, from 1986-2002 were identified. The mean age of presentation was 20 months; 86% were of African-American, Hispanic, or Middle Eastern descent. More than 93% of children had been breastfed; however, 15% had received vitamin D supplementation. Eighty-six percent of those with food histories available were weaned to diets with minimal dairy content after nursing. Serum 25-hydroxyvitamin D was 20.9 +/- 11.5 ng/ml and was less than 15 ng/ml in only 22% of patients. Three representative case histories suggest that dietary calcium intake may play a contributory role in the development of disease; 1 case documents radiographic and biochemical resolution of rachitic abnormalities after calcium treatment, but no vitamin D therapy. Clinicians should be aware that low dietary calcium intake after weaning may result in the development of nutritional rickets, and that attention to calcium intake as well as that of vitamin D is important in the establishment of optimal dietary practices for North American infants and children.
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http://dx.doi.org/10.1210/jc.2002-021935DOI Listing
August 2003

Analysis of continuous glucose monitoring data from non-diabetic and diabetic children: a tale of two algorithms.

Diabetes Technol Ther 2003 ;5(3):375-80

Department of Pediatrics and Children's Clinical Research Center, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

Use of the Medtronic MiniMed Continuous Glucose Monitoring System (CGMS) in non-diabetic children has revealed many low and high sensor glucose (SG) values, suggesting that the original analytical algorithm (Solutions 2.0) might be overreading glycemic excursions. A revised algorithm (Solutions 3.0) was introduced in 2001. Our aim was to compare analyses of the same sensor profiles using both programs. Twenty-five lean, non-diabetic subjects (mean age 14 +/- 4 years) underwent continuous glucose monitoring with CGMS for up to 72 h. Sensor tracings were analyzed with both algorithms and compared. Separate analyses were performed for nocturnal readings (12-6 a.m.). Mean SG values were similar (103 +/- 24 mg/dL for version 2.0 vs. 100 +/- 14 for version 3.0), but the distribution was significantly different: 13.8% of total SG were <70 mg/dL by version 2.0 versus 8.2% by version 3.0 (p < 0.001), and 7.7% of total SG were >150 mg/dL by version 2.0 versus 4.7% by version 3.0 (p = 0.02). Of nocturnal SG values, 25.8% were <70 mg/dL by version 2.0 compared with 17.9% by version 3.0, and 9.4% were >150 mg/dL by version 2.0 compared with 4.0% by version 3.0. In lean non-diabetic children, Solutions 2.0 identified significantly more hypoglycemia and hyperglycemia than Solutions 3.0. Similar analyses in 40 children with type 1 diabetes revealed no significant differences. Solutions 3.0 may be a more useful algorithm for preventing over-reading of low and high SG readings in non-diabetic children, whereas both algorithms give similar results in children with diabetes.
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http://dx.doi.org/10.1089/152091503765691866DOI Listing
August 2003

Rickets in the sunshine?

Nutrition 2002 Jan;18(1):97-9

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http://dx.doi.org/10.1016/s0899-9007(01)00676-1DOI Listing
January 2002