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ABSTRACT: HIV-1 integrase is an important therapeutic target in the fight against HIV/AIDS. Integrase strand transfer inhibitors (INSTIs), which target the enzyme active site, have witnessed clinical success over the past 5 years, but the generation of drug resistance poses challenges to INSTI-based therapies moving forward. Integrase is a dynamic protein, and its ordered multimerization is critical to enzyme activity. The integrase tetramer, bound to viral DNA, interacts with host LEDGF/p75 protein to tether integration to active genes. Allosteric integrase inhibitors (ALLINIs) that compete with LEDGF/p75 for binding to integrase disrupt integrase assembly with viral DNA and allosterically inhibit enzyme function. ALLINIs display steep dose response curves and synergize with INSTIs ex vivo, highlighting this novel inhibitor class for clinical development.
Current opinion in chemical biology 05/2013; · 8.30 Impact Factor
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Lei Feng,
Amit Sharma,
Alison Slaughter,
Nivedita Jena,
Yasuhiro Koh,
Nikolozi Shkriabai,
Ross C Larue,
Pratiq A Patel,
Hiroaki Mitsuya,
Jacques J Kessl,
Alan Engelman,
James R Fuchs, Mamuka Kvaratskhelia
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ABSTRACT: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-LEDGF/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we have elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction, but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead ALLINIs markedly altered the multimerization of IN by promoting aberrant, higher order wild type, but not A128T, IN oligomers. Accordingly, wild type IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors in both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. Taken together, we conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance.
Journal of Biological Chemistry 04/2013; · 4.77 Impact Factor
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Kellie A Jurado,
Hao Wang,
Alison Slaughter,
Lei Feng,
Jacques J Kessl,
Yasuhiro Koh,
Weifeng Wang,
Allison Ballandras-Colas,
Pratiq A Patel,
James R Fuchs, Mamuka Kvaratskhelia,
Alan Engelman
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ABSTRACT: Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development.
Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
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Jocelyn O Eidahl,
Brandon L Crowe,
Justin A North,
Christopher J McKee,
Nikoloz Shkriabai,
Lei Feng,
Matthew Plumb,
Robert L Graham,
Robert J Gorelick,
Sonja Hess,
Michael G Poirier,
Mark P Foster, Mamuka Kvaratskhelia
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ABSTRACT: Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin.
Nucleic Acids Research 02/2013; · 8.03 Impact Factor
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Hao Wang,
Kellie A Jurado,
Xiaolin Wu,
Ming-Chieh Shun,
Xiang Li,
Andrea L Ferris,
Steven J Smith,
Pratiq A Patel,
James R Fuchs,
Peter Cherepanov, Mamuka Kvaratskhelia,
Stephen H Hughes,
Alan Engelman
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ABSTRACT: The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.
Nucleic Acids Research 10/2012; · 8.03 Impact Factor
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ABSTRACT: Human T lymphotropic virus type-1 (HTLV-1) and type 2 (HTLV-2) are closely related human retroviruses, but have unique disease associations. HTLV-1 is the causative agent of an aggressive T-cell leukemia known as adult T-cell leukemia (ATL), HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. HTLV-2 infection has not been clearly associated with any disease condition. Although both viruses can transform T cells in vitro, the HTLV-1 provirus is mainly detected in CD4+ T cells whereas HTLV-2 is mainly detected in CD8+ T cells of infected individuals. HTLV-1 and HTLV-2 encode accessory proteins p30 and p28, respectively, which share partial amino acid homology and are required for viral persistence in vivo. The goal of this study was to identify host proteins interacting with p30 and p28 in order to understand their role in pathogenesis.
Affinity-tag purification coupled with mass spectrometric (MS) analyses revealed 42 and 22 potential interacting cellular partners of p30 and p28, respectively. Of these, only three cellular proteins, protein arginine methyltransferase 5 (PRMT5), hnRNP K and 60 S ribosomal protein L8 were detected in both p30 and p28 fractions. To validate the proteomic results, four interacting proteins were selected for further analyses using immunoblot assays. In full agreement with the MS analysis two cellular proteins REGγ and NEAF-interacting protein 30 (NIP30) selectively interacted with p30 and not with p28; heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) bound to p28 and not to p30; and PRMT5 interacted with both p30 and p28. Further studies demonstrated that reduced levels of PRMT5 resulted in decreased HTLV-2 viral gene expression whereas the viral gene expression of HTLV-1 was unchanged.
The comparisons of p30 and p28 host protein interaction proteome showed striking differences with some degree of overlap. PRMT5, one of the host proteins that interacted with both p30 and p28 differentially affected HTLV-1 and HTLV-2 viral gene expression suggesting that PRMT5 is involved at different stages of HTLV-1 and HTLV-2 biology. These findings suggest that distinct host protein interaction profiles of p30 and p28 could, in part, be responsible for differences in HTLV-1 and HTLV-2 pathobiology. This study provides new avenues of investigation into mechanisms of viral infection, tropism and persistence.
Retrovirology 08/2012; 9:64. · 6.47 Impact Factor
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Ross Larue,
Kushol Gupta,
Christiane Wuensch,
Nikolozi Shkriabai,
Jacques J Kessl,
Eric Danhart,
Lei Feng,
Oliver Taltynov,
Frauke Christ,
Gregory D Van Duyne,
Zeger Debyser,
Mark P Foster, Mamuka Kvaratskhelia
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ABSTRACT: Transportin 3 (TNPO3 or TRN-SR2) has been shown to be an important cellular factor for early steps of lentiviral replication. However, separate studies have implicated distinct mechanisms for TNPO3 either through its interaction with HIV-1 integrase or capsid. Here we have carried out a detailed biophysical characterization of TNPO3 and investigated its interactions with viral proteins. Biophysical analyses including circular dichroism, analytical ultracentrifugation, small-angle x-ray scattering, and homology modeling provide insight into TNPO3 architecture and indicate that it is highly structured and exists in a monomer-dimer equilibrium in solution. In vitro biochemical binding assays argued against meaningful direct interaction between TNPO3 and the capsid cores. Instead, TNPO3 effectively bound to the functional intasome but not to naked viral DNA, suggesting that TNPO3 can directly engage the HIV-1 IN tetramer prebound to the cognate DNA. Mass spectrometry-based protein footprinting and site-directed mutagenesis studies have enabled us to map several interacting amino acids in the HIV-1 IN C-terminal domain and the cargo binding domain of TNPO3. Our findings provide important information for future genetic analysis to better understand the role of TNPO3 and its interacting partners for HIV-1 replication.
Journal of Biological Chemistry 08/2012; 287(41):34044-58. · 4.77 Impact Factor
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Jacques J Kessl,
Nivedita Jena,
Yasuhiro Koh,
Humeyra Taskent-Sezgin,
Alison Slaughter,
Lei Feng,
Suresh de Silva,
Li Wu,
Stuart F J Le Grice,
Alan Engelman,
James R Fuchs, Mamuka Kvaratskhelia
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ABSTRACT: The multifunctional HIV-1 enzyme integrase interacts with viral DNA and its key cellular cofactor LEDGF to effectively integrate the reverse transcript into a host cell chromosome. These interactions are crucial for HIV-1 replication and present attractive targets for antiviral therapy. Recently, 2-(quinolin-3-yl) acetic acid derivatives were reported to selectively inhibit the integrase-LEDGF interaction in vitro and impair HIV-1 replication in infected cells. Here, we show that this class of compounds impairs both integrase-LEDGF binding and LEDGF-independent integrase catalytic activities with similar IC(50) values, defining them as bona fide allosteric inhibitors of integrase function. Furthermore, we show that 2-(quinolin-3-yl) acetic acid derivatives block the formation of the stable synaptic complex between integrase and viral DNA by allosterically stabilizing an inactive multimeric form of integrase. In addition, these compounds inhibit LEDGF binding to the stable synaptic complex. This multimode mechanism of action concordantly results in cooperative inhibition of the concerted integration of viral DNA ends in vitro and HIV-1 replication in cell culture. Our findings, coupled with the fact that high cooperativity of antiviral inhibitors correlates with their increased instantaneous inhibitory potential, an important clinical parameter, argue strongly that improved 2-(quinolin-3-yl) acetic acid derivatives could exhibit desirable clinical properties.
Journal of Biological Chemistry 03/2012; 287(20):16801-11. · 4.77 Impact Factor
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ABSTRACT: A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN-viral DNA and IN-LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN-viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN-LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.
Nucleic Acids Research 07/2011; 39(20):9009-22. · 8.03 Impact Factor
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ABSTRACT: A detailed understanding of how aptamers recognize biological binding partners is of considerable importance in the development of oligonucleotide therapeutics. For antiviral nucleic acid aptamers, current models predict a correlation between broad-spectrum inhibition of viral proteins and suppression of emerging viral resistance, but there is little understanding of how aptamer structures contribute to recognition specificity. We previously established that two independent single-stranded DNA aptamers, R1T and RT1t49(-5), are potent inhibitors of reverse transcriptases (RTs) from diverse branches of the primate lentiviral family, including HIV-1, HIV-2 and SIV(cpz). In contrast, class 1 RNA pseudoknots, such as aptamer T1.1, are specific for RTs from only a few viral clades. Here, we map the binding interfaces of complexes formed between RT and aptamers R1T, RT1t49(-5) and T1.1, using mass spectrometry-based protein footprinting of RT and hydroxyl radical footprinting of the aptamers. These complementary methods reveal that the broad-spectrum aptamers make contacts throughout the primer-template binding cleft of RT. The double-stranded stems of these aptamers closely mimic natural substrates near the RNase H domain, while their binding within the polymerase domain significantly differs from RT substrates. These results inform our perspective on how sustained, broad-spectrum inhibition of RT can be achieved by aptamers.
Nucleic Acids Research 07/2011; 39(18):8237-47. · 8.03 Impact Factor
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ABSTRACT: The DExH protein RNA helicase A (RHA) plays numerous roles in cell physiology, and post-transcriptional activation of gene expression is a major role among them. RHA selectively activates translation of complex cellular and retroviral mRNAs. Although RHA requires interaction with structural features of the 5'-UTR of these target mRNAs, the molecular basis of their translation activation by RHA is poorly understood. RHA contains a conserved ATPase-dependent helicase core that is flanked by two α-β-β-β-α double-stranded RNA-binding domains at the N terminus and repeated arginine-glycine residues at the C terminus. The individual recombinant N-terminal, central helicase, and C-terminal domains were evaluated for their ability to specifically interact with cognate RNAs by in vitro biochemical measurements and mRNA translation assays in cells. The results demonstrate that N-terminal residues confer selective interaction with retroviral and junD target RNAs. Conserved lysine residues in the distal α-helix of the double-stranded RNA-binding domains are necessary to engage structural features of retroviral and junD 5'-UTRs. Exogenous expression of the N terminus coprecipitates junD mRNA and inhibits the translation activity of endogenous RHA. The results indicate that the molecular basis for the activation of translation by RHA is recognition of target mRNA by the N-terminal domain that tethers the ATP-dependent helicase for rearrangement of the complex 5'-UTR.
Journal of Biological Chemistry 02/2011; 286(7):5328-37. · 4.77 Impact Factor
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ABSTRACT: Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.
Journal of Biological Chemistry 01/2011; 286(9):7661-8. · 4.77 Impact Factor
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ABSTRACT: An integrated HIV-1 genomic DNA leads to an infected cell becoming either an active or a latent virus-producing cell. Upon appropriate activation, a latently infected cell can result in production of progeny viruses that spread the infection to uninfected cells. The host proteins influence several steps of HIV-1 infection including formation of the preintegration complex (PIC), a key nucleoprotein intermediate essential for integration of reverse transcribed viral DNA into the chromosome. Much effort has gone into the identification of host proteins contributing to the assembly of functional PICs. Experimental approaches included the use of yeast two-hybrid system, co-immunoprecipitation, affinity tagged HIV-1 viral proteins and in vitro reconstitution of salt-stripped PIC activity. Several host proteins identified using these approaches have been shown to affect HIV-1 replication in cells and influence catalytic activities of recombinant IN in vitro. However, the comprehensive identification and characterization of host proteins associated with HIV-1 PICs of infected cells have been hindered in part by the technical limitation in acquiring sufficient amount of catalytically active PICs. To efficiently identify additional host factors associated with PICs in infected cells, we have developed the following novel approach. The catalytically active PICs from HIV-1-infected CD4+ cells were isolated using biotinylated target DNA, and the proteins selectively co-purifying with PICs have been analyzed by mass spectrometry. This technology enabled us to reveal at least 19 host proteins that are associated with HIV-1 PICs, of which 18 proteins have not been described previously with respect to HIV-1 integration. Physiological functions of the identified proteins range from chromatin organization to protein transport. A detailed characterization of these host proteins could provide new insights into the mechanism of HIV-1 integration and uncover new antiviral targets to block HIV-1 integration.
Retrovirology 01/2010; 7:66. · 6.47 Impact Factor
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ABSTRACT: Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review.
Viruses 12/2009; 1(3):713-36. · 1.50 Impact Factor
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ABSTRACT: HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the small-molecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN.
Molecular pharmacology 08/2009; 76(4):824-32. · 4.53 Impact Factor
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ABSTRACT: Crystallographic studies of the C-terminal DNA polymerase-beta-like domain of full-length human DNA polymerase lambda (fPollambda) suggested that the catalytic cycle might not involve a large protein domain rearrangement as observed with several replicative DNA polymerases and DNA polymerase beta. To examine solution-phase protein conformational changes in fPollambda, which also contains a breast cancer susceptibility gene 1 C-terminal domain and a proline-rich domain at its N-terminus, we used a mass spectrometry-based protein footprinting approach. In parallel experiments, surface accessibility maps for Arg residues were compared for the free fPollambda versus the binary complex of enzyme*gapped DNA and the ternary complex of enzyme*gapped DNA*dNTP (2'-deoxynucleotide triphosphate). These experiments suggested that fPollambda does not undergo major conformational changes during the catalysis in the solution phase. Furthermore, the mass spectrometry-based protein footprinting experiments revealed that active site residue R386 was shielded from the surface only in the presence of both a gapped DNA substrate and an incoming nucleotide. Site-directed mutagenesis and pre-steady-state kinetic studies confirmed the importance of R386 for the enzyme activity and indicated the key role for its guanidino group in stabilizing the negative charges of an incoming nucleotide and the leaving pyrophosphate product. We suggest that such interactions could be shared by and important for catalytic functions of other DNA polymerases.
Journal of Molecular Biology 08/2009; 390(3):368-79. · 4.00 Impact Factor
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ABSTRACT: In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation.
Journal of Biological Chemistry 08/2009; 284(36):24213-22. · 4.77 Impact Factor
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ABSTRACT: CD24 is a glycosyl-phosphatidyl-inositol linked glycoprotein expressed in a broad range of cell types and is heavily glycosylated. It has been found to be over expressed in cancers and tumors and is also a costimulatory molecule. Therefore, this study was carried out to define the structures of the carbohydrates associated with the CD24 recombinant protein. The CD24 glycoprotein's oligosaccharides were released by chemical and enzymatic means prior to being analyzed by MALDI-TOF-MS. The results obtained showed that CD24 is both N- and O-glycosylated. The major oligosaccharides were found to be Neu5Acα-2,3/6Galβ-1,3GalNAc, NeuAc(2)Gal β-1,3GalNAc(1) (O-glycans), GalNAc(2)GlcNAc(2)Man(3)Fuc(1), Gal(1)GalNAc(2)GlcNAc(2)Man(3)Fuc(1), and Gal(2)GalNAc(2)GlcNAc(2)Man(3)Fuc(1) (N-glycans). The results showed that Neu5Acα-2,3/6Galβ-1,3GalNAc (sialyl-tumor antigen, sT), a cancer-associated carbohydrate, was the most abundant glycan associated with CD24. This result raised the intriguing possibility that CD24 may be a major carrier of the sialyl-T abundantly found in cancer cells.
American journal of biomedical sciences. 01/2009; 1(1):1-11.
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ABSTRACT: We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein-protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino acids selectively shielded from modification in the protein-protein complex. The current report focuses on probing interactions between full-length HIV-1 integrase and its principal cellular partner lens epithelium-derived growth factor. This method has a generic application and is particularly attractive for studying large protein-protein interactions that are less amenable for crystallographic or NMR analysis.
Methods 12/2008; 47(4):304-7. · 4.01 Impact Factor
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Michaela Wendeler,
Hsiu-Fang Lee,
Alun Bermingham,
Jennifer T Miller,
Oleg Chertov,
Marion K Bona,
Noel S Baichoo,
Maryam Ehteshami,
John Beutler,
Barry R O'Keefe,
Matthias Götte, Mamuka Kvaratskhelia,
Stuart Le Grice
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ABSTRACT: High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4 H-cyclohepta[ b]thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT). A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor beta-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Site-directed mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or non-natural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, divalent metal-chelating inhibitors that have been reported.
ACS Chemical Biology 11/2008; 3(10):635-44. · 6.45 Impact Factor
