Timothy J Cardozo, M.D., Ph.D.

Quantitative assessment of masking of neutralization epitopes in HIV-1
Agarwal, Alpna; Hioe, Catarina E; Swetnam, James; Zolla-Pazner, Susan; Cardozo, Timothy
2011 Sep 9;29(39):6736-6741, Vaccine
Despite the frequent observation of masking of HIV-1 neutralization epitopes, its extent has not been previously systematically assessed either for multiple epitopes presented by individual viruses or for individual epitopes across multiple viral strains. Using a recently developed method to identify amino acid sequence motifs required for recognition by HIV-1-neutralizing monoclonal antibodies (mAbs), we visualized the patterns of masking of specific epitopes targeted by mAbs in a diverse panel of HIV-1 isolates. We also calculated a specific masking intensity score for each virus based on the observed neutralization activity of mAbs against the epitopes in the virus. Finally, we combined these data with estimates of the conservation of each mAb-targeted epitope in circulating HIV-1 strains to estimate the effective neutralization potential (E(N)) for each mAb. Focusing on the V3 loop of gp120 as a prototype neutralization domain, we found that the V3 loop epitope targeted by mAb 2219 is one of the least masked mAbs and it has the highest E(N). Interestingly, although the V3 loop epitope targeted by mAb 3074 is present in over 87% of all viruses, it is 82.2% masked, so its E(N) is lower than that for mAb 2219. Notably, 50% of the viruses that mAb 3074 is able to neutralize are classified as subtype C viruses, while 70% or more of the viruses neutralized by mAbs 2219, 2557 or 447-52D are classified as subtype B. Thus, neutralization epitopes (in this case, in the V3 loop) have differential patterns of masking and also display distinct patterns of distribution among circulating HIV-1 viruses. Both factors combine to contribute to the practical vaccine value of any single epitope/mAb. Here we have developed a quantitative score for this value. These results have important implications for rational design of vaccines designed to induce neutralizing Abs by revealing epitopes that are minimally masked and maximally reactive with neutralizing Abs
— id: 137058, year: 2011, vol: 29, page: 6736, stat: Journal Article,

The Architecture of CopA from Archeaoglobus fulgidus Studied by Cryo-Electron Microscopy and Computational Docking
Allen, Gregory S; Wu, Chen-Chou; Cardozo, Tim; Stokes, David L
2011 Sep 7;19(9):1219-1232, Structure
CopA uses ATP to pump Cu(+) across cell membranes. X-ray crystallography has defined atomic structures of several related P-type ATPases. We have determined a structure of CopA at 10 A resolution by cryo-electron microscopy of a new crystal form and used computational molecular docking to study the interactions between the N-terminal metal-binding domain (NMBD) and other elements of the molecule. We found that the shorter-chain lipids used to produce these crystals are associated with movements of the cytoplasmic domains, with a novel dimer interface and with disordering of the NMBD, thus offering evidence for the transience of its interaction with the other cytoplasmic domains. Docking identified a binding site that matched the location of the NMBD in our previous structure by cryo-electron microscopy, allowing a more detailed view of its binding configuration and further support for its role in autoinhibition
— id: 137070, year: 2011, vol: 19, page: 1219, stat: Journal Article,

Tagetitoxin inhibits RNA polymerase through trapping of the trigger loop
Artsimovitch I.; Svetlov V.; Nemetski S.M.; Epshtein V.; Cardozo T.; Nudler E.
2011 ;286(46):40395-40400, Journal of biological chemistry
Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the beta' subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a 'slow' enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics. 2011 by The American Society for Biochemistry and Molecular Biology, Inc
— id: 142055, year: 2011, vol: 286, page: 40395, stat: Journal Article,

Feature Article: From the Cover: An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway
Gonsalves, Foster C; Klein, Keren; Carson, Brittany B; Katz, Shauna; Ekas, Laura A; Evans, Steve; Nagourney, Robert; Cardozo, Timothy; Brown, Anthony M C; Dasgupta, Ramanuj
2011 Apr 12;108(15):5954-5963, Proceedings of the National Academy of Sciences of the United States of America
Misregulated beta-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of beta-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear beta-catenin. We show that these inhibitors efficiently block Wnt/beta-catenin-induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling
— id: 130910, year: 2011, vol: 108, page: 5954, stat: Journal Article,

Human Anti-V3 HIV-1 Monoclonal Antibodies Encoded by the VH5-51/VL Lambda Genes Define a Conserved Antigenic Structure
Gorny, Miroslaw K; Sampson, Jared; Li, Huiguang; Jiang, Xunqing; Totrov, Maxim; Wang, Xiao-Hong; Williams, Constance; O'Neal, Timothy; Volsky, Barbara; Li, Liuzhe; Cardozo, Timothy; Nyambi, Phillipe; Zolla-Pazner, Susan; Kong, Xiang-Peng
2011 ;6(12):e27780-e27780, PLoS ONE
Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs
— id: 146267, year: 2011, vol: 6, page: e27780, stat: Journal Article,

Recovering Protein-Protein and Domain-Domain Interactions from Aggregation of IP-MS Proteomics of Coregulator Complexes
Mazloom, Amin R; Dannenfelser, Ruth; Clark, Neil R; Grigoryan, Arsen V; Linder, Kathryn M; Cardozo, Timothy J; Bond, Julia C; Boran, Aislyn D W; Iyengar, Ravi; Malovannaya, Anna; Lanz, Rainer B; Ma'ayan, Avi
2011 Dec;7(12):e1002319-e1002319, PLoS Computational Biology
Coregulator proteins (CoRegs) are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP) followed by mass spectrometry (MS) applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/
— id: 149983, year: 2011, vol: 7, page: e1002319, stat: Journal Article,

Indirect Detection of an Epitope-Specific Response to HIV-1 gp120 Immunization in Human Subjects
Shmelkov, Evgeny; Nadas, Arthur; Swetnam, James; Zolla-Pazner, Susan; Cardozo, Timothy
2011 ;6(11):e27279-e27279, PLoS ONE
A specific response of human serum neutralizing antibodies (nAb) to a conformational epitope as a result of vaccination of human subjects with the surface envelope glycoprotein (gp120) of HIV-1 has not previously been documented. Here, we used computational analysis to assess the epitope-specific responses of human subjects, which were immunized with recombinant gp120 immunogens in the VAX003 and VAX004 clinical trials. Our computational methodology-a variation of sieve analysis-compares the occurrence of specific nAb targeted conformational 3D epitopes on viruses from infected individuals who received vaccination to the occurrence of matched epitopes in the viruses infecting placebo subjects. We specifically studied seven crystallographically defined nAb targeted conformational epitopes in the V3 loop, an immunogenic region of gp120. Of the six epitopes present in the immunogens and targeted by known monoclonal neutralizing antibodies, only the one targeted by the anti-V3 nAb 2219 exhibited a significant reduction in occurrence in vaccinated subjects compared to the placebo group. This difference occurred only in the VAX003 Thailand cohort. No difference was seen between vaccinated and placebo groups for the occurrence of an epitope that was not present in the immunogen. Thus, it can be theorized that a specific 2219-like human neutralizing antibody immune response to AIDSVAX immunization occurred in the VAX003 cohort, and that this response protected subjects from a narrow subset of HIV-1 viruses circulating in Thailand in the 1990s and bearing the conformational epitope targeted by the neutralizing antibody 2219
— id: 141491, year: 2011, vol: 6, page: e27279, stat: Journal Article,

"Corrigendum to Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold"" [Virology 351 (2010) 513-523]"
Totrov M.; Jiang X.; Kong X.-P.; Cohen S.; Krachmarov C.; Salomon A.; Williams C.; Seaman M.S.; Abagyan R.; Cardozo T.; Gorny M.K.; Wang S.; Lu S.; Pinter A.; Zolla-Pazner S.
2011 ;409(2):360-360, Virology
— id: 119242, year: 2011, vol: 409, page: 360, stat: Journal Article,

V2-reactive antibodies in RV144 vaccinees' plasma
Zolla-Fazner S.; Cardozo T.; De Camp A.; Haynes B.; Kim J.; Kong X.; Michael N.; Rerks-Ngarm S.; Williams C.
2011 ;27(10):A21-A21, AIDS research & human retroviruses
Background: Initial studies suggested that V2 antibodies (Abs) played a protective role in RV144. Filot experiments were needed to quantify and characterize anti-V2 Abs in plasma of RV144 participants. Methods: One hundred coded RV144 pilot study specimens were analyzed by ELISA using plasma obtained before immunization (visit 1), and 2 and 28 weeks after the last immunizing dose (visits 8 and 9, respectively). Results were decoded after submission and analysis of data. Results: [a] At visit 1, 0/20 placebo and 3/80 vaccinee specimens diluted 1:20 reacted with gp120JR-FL. At visit 8, 0/20 and 80/80 were reactive. Flasma were not reactive vs. bovine serum albumin. [b] At visit 1, 0/20 placebo and 1/80 vaccinee specimens diluted 1:20 reacted with a V1V2HxB2-gp70 fusion protein. At visit 8, 1/20 and 76/80 (95%) were reactive. [c] Reactivity was tested against four 21-mer linear V2 peptides spanning the a4/?7 binding motif, chosen to represent: the most polar sequence found in V2 loops with 38 amino acids ('polar-38'); the most common sequence of V2s with 40 AAs ('common-40'); the most polar sequence in V2 loops with 40 AAs ('polar-40'); and the consensus sequence of V2 loops with 40 AAs ('consensus-40'). Activity in plasma diluted 1:100 showed differential reactivity with the peptides: 'polar-38' & 'polar-40'>'consensus'>'common- 40'. For visit 8, 1/20 placebo and 47/80 (59%) vaccine specimens were reactive with 'polar-38'. Shorter linear peptides were poorly reactive. Notably, by visit 9, the reactivity to gp120_JR-FL, V1V2_HxB2-gp70, and all V2 peptides was markedly reduced in all vaccinees' plasmas. Conclusion: Anti-V2 Abs were elicited by the RV144 immunization protocol. Reactivity was highest 2 weeks after the last boost, and was significantly diminished 26 weeks later. V2-spe- cific reactivity was demonstrable with a fusion protein containing the entire V1V2 loop and with 21-mer linear V2 peptides spanning the a4/?7 binding motif
— id: 139488, year: 2011, vol: 27, page: A21, stat: Journal Article,

Cross-Clade HIV-1 Neutralizing Antibodies Induced with V3-Scaffold Protein Immunogens following Priming with gp120 DNA
Zolla-Pazner, Susan; Kong, X-P; Jiang, Xunqing; Cardozo, Timothy; Nadas, Arthur; Cohen, Sandra; Totrov, Maxim; Seaman, Michael S; Wang, Shixia; Lu, Shan
2011 Oct;85(19):9887-9898, Journal of virology
The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3(C)-CTB), or with double combinations of V3-CTB immunogens that included V3(C)-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity
— id: 137442, year: 2011, vol: 85, page: 9887, stat: Journal Article,

Quantitative Assessment of HIV-1 Neutralization Epitope Masking
Agarwal, A.; Swetnam, J.; Zolla-Pazner, S.; Cardozo, T.
2010 OCT ;26(10):A74-A74, AIDS research & human retroviruses
— id: 117318, year: 2010, vol: 26, page: A74, stat: Journal Article,

Map of broad and narrow neutralization in the V3 loop crown
Almond D.; Kong X.; Zolla-Pazner S.; Cardozo T.
2010 ;26(10):A27-A28, AIDS research & human retroviruses
Background: Sequence variability of the V3 loop crown has often been considered an impediment to eliciting broadly neutralization antibodies targeted to this region. Our work maps contacts between human anti-V3 monoclonal antibodies (mAbs) and the V3 crown in an attempt to make a structural distinction between the sites targeted by broadly as opposed to narrowly neutralizing mAbs. Methods: The 3D structure of the 16-residue V3 crown bound to anti-V3 mAb 2219 was divided into 4 regions (stem, hydrophilic patch, hydrophobic patch and turn) based on previously published analyses (Almond et. al., 2010). Known mAb/V3 contacts from Jiang et al., 2010 and others were then mapped onto this structure and compared to measurements of neutralization breadth by the respective mAbs in infectivity assays. Results: mAbs that entirely engage the less sequence variable zones of the V3 loop (the stem, turn and hydrophobic zones) tend to be more broadly neutralizing than mAbs that contact at least one side-chain in the most variable region of V3 (the hydrophilic zone). The contact in the highly variable region appears to narrow the Ab reactivity regardless of how many other contacts are formed with V3 backbone atoms or with side-chains in the conserved regions. Conclusion: Broadly neutralizing mAbs are targeted to the structurally conserved region, and they avoid contact with the highly sequence-variable zone. More narrowly neutralizing Abs make at least one side-chain contact within the highly-sequence variable zone: these latter mAbs are vulnerable to a single escape mutation at that side-chain/amino acid location, and indeed these side-chains are frequently mutated in circulating viruses. This knowledge could be exploited for HIV vaccine design by designing immunogens that immunologically silence the variable zone in the V3 loop
— id: 114523, year: 2010, vol: 26, page: A27, stat: Journal Article,

Structural conservation predominates over sequence variability in the crown of HIV type 1's V3 loop
Almond, David; Kimura, Tetsuya; Kong, XiangPeng; Swetnam, James; Zolla-Pazner, Susan; Cardozo, Timothy
2010 Jun;26(6):717-723, AIDS research & human retroviruses
The diversity of HIV-1 is a confounding problem for vaccine design, as the human immune response appears to favor poor or strain-specific responses to any given HIV-1 virus strain. A significant portion of this diversity is manifested as sequence variability in the loops of HIV-1's surface envelope glycoprotein. Here we show that the most variable sequence positions in the third variable (V3) loop crown cluster to a small zone on the surface of one face of the V3 loop ss-hairpin conformation. These results provide a novel visualization of the gp120 V3 loop, specifically demonstrating a surprising preponderance of conserved three-dimensional structure in a highly sequence-variable region. From a structural point of view, there appears to be less diversity in this region of the HIV-1 'principle neutralizing domain' than previously appreciated
— id: 110086, year: 2010, vol: 26, page: 717, stat: Journal Article,

Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery
Band, Philip A; Blais, Steven; Neubert, Thomas A; Cardozo, Timothy J; Ichtchenko, Konstantin
2010 May;71(1):62-73, Protein expression & purification
Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1ad(ek) and BoNT/A1ad(tev) are full length derivatives rendered atoxic through double point mutations in the LC protease (E(224)>A; Y(366)>A). DeltaLC-peptide-BoNT/A(tev) and DeltaLC-GFP-BoNT/A(tev) are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HC) influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm
— id: 107925, year: 2010, vol: 71, page: 62, stat: Journal Article,

Engineered immunogen presenting an epitope recognized by a neutralizing mAb elicits mammalian serum that recapitulate the mAb's specificity
Cardozo T.; Kong X.; Totrov M.; Wang S.; Lu S.; Gorny M.; Pinter A.; Seaman M.; Zolla-Pazner S.
2010 ;26(10):A26-A27, AIDS research & human retroviruses
Background: To exploit the promising properties of cross-strain neutralizing monoclonal antibodies (mAbs), immunogens should elicit polyclonal Ab responses in mammals that mimic the reactivity of these mAbs. To demonstrate the feasibility of this approach, the epitope recognized by the anti-V3 loop mAb 3074- which is present in approximately 90% of circulating HIV-1 viruses-was use as a template for immunogen design. Methods: A V3 loop-cholera toxin B fusion protein (V3₃₀₇₄-CTB) was designed to eliminate the epitopes targeted by several anti-V3 loop mAbs while preserving the epitope targeted by mAb 3074. New Zealand White rabbits were primed with codon-optimized clade C gp120 DNA and then boosted with V3₃₀₇₄-CTB. Anti-V3 mAbs and rabbit immune sera were assessed for neutralizing activity against (a) V3 chimeric psVs infecting U87 CD4 + CCR5 + cells, (b) primary isolates infecting TZM-bl cells, (c) Tier 1 and (d) Tier 2 psVs infecting TZM.bl cells. Results: V33074-CTB bound specifically to mAb 3074 but not to several other anti-V3 mAbs. A psV bearing the V3₃₀₇₄-designed sequence was neutralized only by mAb 3074. Immune sera elicited in rabbits using V3₃₀₇₄-CTB demonstrated 50% neutralization of (a) 7/7 V3 chimeric psVs carrying the V3 loops of clades A1, AG, AE, B, C, F, and H, (b) 3/10 primary isolates from clades A, AG and B, (c) 4/4 Tier 1 viruses, and (d) 4/14 Tier 2 viruses from clades B and C. Little neutralizing activity was seen in the immune sera against a V3 chimeric psV lacking the 3074 epitope, and the only three Tier 2 clade C viruses neutralized by mAb 3074 were also the only three Tier 2 Clade C viruses neutralized by the serum of the best responder. Conclusion: Our results demonstrate that V3₃₀₇₄-CTB elicited a polyclonal, cross-strain neutralizing Ab response in mammalian serum mirroring the specificity of 3074-the mAb used as a template for immunogen design
— id: 114522, year: 2010, vol: 26, page: A26, stat: Journal Article,

Characterization of a dominant-active STAT that promotes tumorigenesis in Drosophila
Ekas, Laura A; Cardozo, Timothy J; Flaherty, Maria Sol; McMillan, Elizabeth A; Gonsalves, Foster C; Bach, Erika A
2010 Aug 15;344(2):621-636, Developmental biology (Orlando)
Little is known about the molecular mechanisms by which STAT proteins promote tumorigenesis. Drosophila is an ideal system for investigating this issue, as there is a single STAT (Stat92E), and its hyperactivation causes overgrowths resembling human tumors. Here we report the first identification of a dominant-active Stat92E protein, Stat92E(DeltaNDeltaC), which lacks both N- and C-termini. Mis-expression of Stat92E(DeltaNDeltaC)in vivo causes melanotic tumors, while in vitro it transactivates a Stat92E-luciferase reporter in the absence of stimulation. These gain-of-function phenotypes require phosphorylation of Y(711) and dimer formation with full-length Stat92E. Furthermore, a single point mutation, an R(442P) substitution in the DNA-binding domain, abolishes Stat92E function. Recombinant Stat92E(R442P) translocates to the nucleus following activation but fails to function in all assays tested. Interestingly, R(442) is conserved in most STATs in higher organisms, suggesting conservation of function. Modeling of Stat92E indicates that R(442) may contact the minor groove of DNA via invariant TC bases in the consensus binding element bound by all STAT proteins. We conclude that the N- and C- termini function unexpectedly in negatively regulating Stat92E activity, possibly by decreasing dimer dephosphorylation or increasing stability of DNA interaction, and that Stat92E(R442) has a nuclear function by altering dimer:DNA binding
— id: 111584, year: 2010, vol: 344, page: 621, stat: Journal Article,

Nuclear receptor engineering based on novel structure activity relationships revealed by farnesyl pyrophosphate
Goyanka, Ritu; Das, Sharmistha; Samuels, Herbert H; Cardozo, Timothy
2010 Nov;23(11):809-815, Protein engineering, design & selection : PEDS
Nuclear receptors (NRs) comprise the second largest protein family targeted by currently available drugs, acting via specific ligand interactions within the ligand binding domain (LBD). Recently, farnesyl pyrophosphate (FPP) was shown to be a unique promiscuous NR ligand, activating a subset of NR family members and inhibiting wound healing in skin. The current study aimed at visualizing the unique basis of FPP interaction with multiple receptors in order to identify general structure-activity relationships that operate across the NR family. Docking of FPP to the 3D structures of the LBDs of a diverse set of NRs consistently revealed an electrostatic FPP pyrophosphate contact with an NR arginine conserved in the NR family, a hydrophobic farnesyl contact with NR helix-12 and a ligand binding pocket volume between 300 and 430 A(3) as the minimal requirements for FPP activation of any NR. Lack of any of these structural features appears to render a given NR resistant to FPP activation. We used these structure-activity relationships to rationally design and successfully engineer several mutant human estrogen receptors that retain responsiveness to estradiol but no longer respond to FPP
— id: 113803, year: 2010, vol: 23, page: 809, stat: Journal Article,

Conserved structural elements in the V3 crown of HIV-1 gp120
Jiang, Xunqing; Burke, Valicia; Totrov, Maxim; Williams, Constance; Cardozo, Timothy; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kong, Xiang-Peng
2010 Aug;17(8):955-961, Nature structural & molecular biology
Binding of the third variable region (V3) of the HIV-1 envelope glycoprotein gp120 to the cell-surface coreceptors CCR5 or CXCR4 during viral entry suggests that there are conserved structural elements in this sequence-variable region. These conserved elements could serve as epitopes to be targeted by a vaccine against HIV-1. Here we perform a systematic structural analysis of representative human anti-V3 monoclonal antibodies in complex with V3 peptides, revealing that the crown of V3 has four conserved structural elements: an arch, a band, a hydrophobic core and the peptide backbone. These are either unaffected by or are subject to minimal sequence variation. As these regions are targeted by cross-clade neutralizing human antibodies, they provide a blueprint for the design of vaccine immunogens that could elicit broadly cross-reactive protective antibodies
— id: 111542, year: 2010, vol: 17, page: 955, stat: Journal Article,

Computational profiling the epitope-specific human neutralizing antibodies elicited in the AIDSVAX clinical trials
Shmelkov, E.; Nadas, A.; Swetnam, J.; Zolla-Pazner, S.; Cardozo, T.
2010 OCT ;26(10):A44-A44, AIDS research & human retroviruses
— id: 117314, year: 2010, vol: 26, page: A44, stat: Journal Article,

Comparative Magnitude of Cross-Strain Conservation of HIV Variable Loop Neutralization Epitopes
Swetnam, James; Shmelkov, Evgeny; Zolla-Pazner, Susan; Cardozo, Timothy
2010 ;5(12):e15994-e15994, PLoS ONE
Although the sequence variable loops of the human immunodeficiency virus' (HIV-1) surface envelope glycoprotein (gp120) can exhibit good immunogenicity, characterizing conserved (invariant) cross-strain neutralization epitopes within these loops has proven difficult. We recently developed a method to derive sensitive and specific signature motifs for the three-dimensional (3D) shapes of the HIV-1 neutralization epitopes in the third variable (V3) loop of gp120 that are recognized by human monoclonal antibodies (mAbs). We used the signature motif method to estimate the conservation of these epitopes across circulating worldwide HIV-1 strains. The epitope targeted by the anti-V3 loop neutralizing mAb 3074 is present in 87% of circulating strains, distributed nearly evenly among all subtypes. The results for other anti-V3 Abs are: 3791, present in 63% of primarily non-B subtypes; 2219, present in 56% of strains across all subtypes; 2557, present in 52% across all subtypes; 447-52D, present in 11% of primarily subtype B strains; 537-10D, present in 9% of primarily subtype B strains; and 268-D, present in 5% of primarily subtype B strains. The estimates correlate with in vitro tests of these mAbs against diverse viral panels. The mAb 3074 thus targets an epitope that is nearly completely conserved among circulating HIV-1 strains, demonstrating the presence of an invariant structure hidden in the dynamic and sequence-variable V3 loop in gp120. Since some variable loop regions are naturally immunogenic, designing immunogens to mimic their conserved epitopes may be a promising vaccine discovery approach. Our results suggest one way to quantify and compare the magnitude of the conservation
— id: 117360, year: 2010, vol: 5, page: e15994, stat: Journal Article,

Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold
Totrov, Maxim; Jiang, Xunqing; Kong, Xiang-Peng; Cohen, Sandra; Krachmarov, Chavdar; Salomon, Aidy; Williams, Constance; Seaman, Michael S; Abagyan, Ruben; Cardozo, Timothy; Gorny, Miroslaw K; Wang, Shixia; Lu, Shan; Pinter, Abraham; Zolla-Pazner, Susan
2010 Sep 30;405(2):513-523, Virology
V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response
— id: 133786, year: 2010, vol: 405, page: 513, stat: Journal Article,

Structural Determinants of PERK Inhibitor Potency and Selectivity
Wang, Hong; Blais, Jaime; Ron, David; Cardozo, Timothy
2010 Dec;76(6):480-495, Chemical biology & drug design
The unfolded protein response (UPR) is a coordinated program that promotes cell survival under conditions of endoplasmic reticulum stress and is required in tumor progression as well. To date, no specific small molecule inhibitor targeting this pathway has been identified. Pancreatic endoplasmic reticulum kinase (PERK), one of the UPR transducers, is an eIF2alpha kinase. Compromising PERK function inhibits tumor growth in mice, suggesting that PERK may be a cancer drug target, but identifying a specific inhibitor of any kinase is challenging. The goal of this study was to identify some pair-wise receptor-ligand atomic contacts that confer selective PERK inhibition. Compounds selectively inhibiting PERK-mediated phosphorylation in vitro were identified using an initial virtual library screen, followed by structure-activity hypothesis testing. The most potent PERK selective inhibitors utilize three specific kinase active site contacts that, when absent from chemically similar compounds, abrogates the inhibition: (i) a strong van der Waals contact with PERK residue Met7, (ii) interactions with the N-terminal portion of the activation loop, and (iii) groups providing electrostatic complementarity to Asp144. Interestingly, the activation loop contact is required for PERK selectivity to emerge. Understanding these structure-activity relationships may accelerate rational PERK inhibitor design
— id: 114511, year: 2010, vol: 76, page: 480, stat: Journal Article,

Analysis of neutralizing antibody responses induced by gp120 DNA prime followed by monovalent or polyvalent V3 epitope boost
Wang, S.; Lu, S.; Kong, X.; Cardozo, T.; Cohen, S.; Jiang, X.; Totrov, M.; Pinter, A.; Krachmarov, C.; Seaman, M.; Zolla-Pazner, S.
2010 OCT ;26(10):A54-A54, AIDS research & human retroviruses
— id: 117315, year: 2010, vol: 26, page: A54, stat: Journal Article,

Structure-function relationships of HIV-1 envelope sequence-variable regions refocus vaccine design
Zolla-Pazner, Susan; Cardozo, Timothy
2010 Jul;10(7):527-535, Nature reviews. Immunology
One of the main challenges of developing an HIV-1 vaccine lies in eliciting immune responses that can overcome the antigenic variability exhibited by HIV. Most HIV-1 vaccine development has focused on inducing immunity to conserved regions of the HIV-1 envelope. However, new studies of the sequence-variable regions of the HIV-1 gp120 envelope glycoprotein have shown that there are conserved immunological and structural features in these regions. Recombinant immunogens that include these features may provide the means to address the antigenic diversity of HIV-1 and induce protective antibodies that can prevent infection with HIV-1
— id: 110664, year: 2010, vol: 10, page: 527, stat: Journal Article,

Sequence variability in the crown of the V3 loop of the HIV-1 envelope is clustered within a small 3D structural zone
Almond, D; Kimura, T; Kong, X; Swetnam, J; Zolla-Pazner, S; Cardozo, T
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105709, year: 2009, vol: 6, page: 115, stat: Journal Article,

Worldwide distribution of HIV type 1 epitopes recognized by human anti-V3 monoclonal antibodies
Cardozo, Timothy; Swetnam, James; Pinter, Abraham; Krachmarov, Chavdar; Nadas, Arthur; Almond, David; Zolla-Pazner, Susan
2009 Apr;25(4):441-450, AIDS research & human retroviruses
Epitopes, also known as antigenic determinants, are small clusters of specific atoms within macromolecules that are recognized by the immune system. Such epitopes can be targeted with vaccines designed to protect against specific pathogens. The third variable loop (V3 loop) of the HIV-1 pathogen's gp120 surface envelope glycoprotein can be a highly sensitive neutralization target. We derived sequence motifs for the V3 loop epitopes recognized by the human monoclonal antibodies (mAbs) 447-52D and 2219. Searching the HIV database for the occurrence of each epitope motif in worldwide viruses and correcting the results based on published WHO epidemiology reveal that the 447-52D epitope we defined occurs in 13% of viruses infecting patients worldwide: 79% of subtype B viruses, 1% of subtype C viruses, and 7% of subtype A/AG sequences. In contrast, the epitope we characterized for human anti-V3 mAb 2219 is present in 30% of worldwide isolates but is evenly distributed across the known HIV-1 subtypes: 48% of subtype B strains, 40% of subtype C, and 18% of subtype A/AG. Various assays confirmed that the epitopes corresponding to these motifs, when expressed in the SF162 Env backbone, were sensitively and specifically neutralized by the respective mAbs. The method described here is capable of accurately determining the worldwide occurrence and subtype distribution of any crystallographically resolved HIV-1 epitope recognized by a neutralizing antibody, which could be useful for multivalent vaccine design. More importantly, these calculations demonstrate that globally relevant, structurally conserved epitopes are present in the sequence variable V3 loop
— id: 99293, year: 2009, vol: 25, page: 441, stat: Journal Article,

Worldwide epitope prevalence of crystallographically resolved anti-V3 antibodies
Swetnam, J; Zolla-Pazner, S; Cardozo, TJ
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105710, year: 2009, vol: 6, page: 115, stat: Journal Article,

Structure-guided design and immunological characterization of immunogen constructs presenting the HIV-1 gp120 V3 loop on a CTB scaffold
Totrov, M; Jiang, X; Kong, X; Cohen, S; Krachmarov, C; Williams, C; Cardozo, T; Gorny, M; Wang, S; Lu, S; Pinter, A; Zolla-Pazner, S
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105707, year: 2009, vol: 6, page: 115, stat: Journal Article,

Induction of cross-clade neutralizing antibodies with a prime/boost vaccine strategy focused on a neutralizing epitope
Zolla-Pazner, S; Kong, X; Cardozo, T; Hioe, C; Cohen, S; Jiang, X; Gorny, MK; Totrov, M; Pinter, A; Krachmarov, C; Seaman, MS; Wang, S; Lu, S
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105699, year: 2009, vol: 6, page: 115, stat: Journal Article,

Control of chromosome stability by the beta-TrCP-REST-Mad2 axis
Guardavaccaro, Daniele; Frescas, David; Dorrello, N Valerio; Peschiaroli, Angelo; Multani, Asha S; Cardozo, Timothy; Lasorella, Anna; Iavarone, Antonio; Chang, Sandy; Hernando, Eva; Pagano, Michele
2008 Mar 20;452(7185):365-369, Nature
REST/NRSF (repressor-element-1-silencing transcription factor/neuron-restrictive silencing factor) negatively regulates the transcription of genes containing RE1 sites. REST is expressed in non-neuronal cells and stem/progenitor neuronal cells, in which it inhibits the expression of neuron-specific genes. Overexpression of REST is frequently found in human medulloblastomas and neuroblastomas, in which it is thought to maintain the stem character of tumour cells. Neural stem cells forced to express REST and c-Myc fail to differentiate and give rise to tumours in the mouse cerebellum. Expression of a splice variant of REST that lacks the carboxy terminus has been associated with neuronal tumours and small-cell lung carcinomas, and a frameshift mutant (REST-FS), which is also truncated at the C terminus, has oncogenic properties. Here we show, by using an unbiased screen, that REST is an interactor of the F-box protein beta-TrCP. REST is degraded by means of the ubiquitin ligase SCF(beta-TrCP) during the G2 phase of the cell cycle to allow transcriptional derepression of Mad2, an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind beta-TrCP, inhibited Mad2 expression and resulted in a phenotype analogous to that observed in Mad2(+/-) cells. In particular, we observed defects that were consistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chromosome bridges and mis-segregation in anaphase, tetraploidy, and faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant, which does not bind beta-TrCP. Thus, SCF(beta-TrCP)-dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis. The high levels of REST or its truncated variants found in certain human tumours may contribute to cellular transformation by promoting genomic instability
— id: 78365, year: 2008, vol: 452, page: 365, stat: Journal Article,

Aldolase provides an unusual binding site for thrombospondin-related anonymous protein in the invasion machinery of the malaria parasite
Bosch, Jurgen; Buscaglia, Carlos A; Krumm, Brian; Ingason, Bjarni P; Lucas, Robert; Roach, Claudia; Cardozo, Timothy; Nussenzweig, Victor; Hol, Wim G J
2007 Apr 24;104(17):7015-7020, Proceedings of the National Academy of Sciences of the United States of America
An actomyosin motor located underneath the plasma membrane drives motility and host-cell invasion of apicomplexan parasites such as Plasmodium falciparum and Plasmodium vivax, the causative agents of malaria. Aldolase connects the motor actin filaments to transmembrane adhesive proteins of the thrombospondin-related anonymous protein (TRAP) family and transduces the motor force across the parasite surface. The TRAP-aldolase interaction is a distinctive and critical trait of host hepatocyte invasion by Plasmodium sporozoites, with a likely similar interaction crucial for erythrocyte invasion by merozoites. Here, we describe 2.4-A and 2.7-A structures of P. falciparum aldolase (PfAldo) obtained from crystals grown in the presence of the C-terminal hexapeptide of TRAP from Plasmodium berghei. The indole ring of the critical penultimate Trp-residue of TRAP fits snugly into a newly formed hydrophobic pocket, which is exclusively delimited by hydrophilic residues: two arginines, one glutamate, and one glutamine. Comparison with the unliganded PfAldo structure shows that the two arginines adopt new side-chain rotamers, whereas a 25-residue subdomain, forming a helix-loop-helix unit, shifts upon binding the TRAP-tail. The structural data are in agreement with decreased TRAP binding after mutagenesis of PfAldo residues in and near the induced TRAP-binding pocket. Remarkably, the TRAP- and actin-binding sites of PfAldo seem to overlap, suggesting that both the plasticity of the aldolase active-site region and the multimeric nature of the enzyme are crucial for its intriguing nonenzymatic function in the invasion machinery of the malaria parasite
— id: 78751, year: 2007, vol: 104, page: 7015, stat: Journal Article,

Modeling the interaction between aldolase and the thrombospondin-related anonymous protein, a key connection of the malaria parasite invasion machinery
Buscaglia, Carlos A; Hol, Wim G J; Nussenzweig, Victor; Cardozo, Timothy
2007 Feb 15;66(3):528-537, Proteins
A complex molecular motor empowers substrate-dependent motility and host cell invasion in malaria parasites. The interaction between aldolase and the transmembrane adhesin thrombospondin-related anonymous protein (TRAP) transduces the motor force across the parasite surface. Here, we analyzed this interaction by using state-of-the-art flexible docking. Besides algorithms to account for induced fit in the side-chains of the Plasmodium falciparum aldolase (PfAldo) structure, we used additional in silico receptors modeled upon crystallographic structures of evolutionarily related aldolases to incorporate enzyme backbone flexibility, and to overcome structure inaccuracies due to the relatively low resolution (3.0 A) of the genuine PfAldo structure. Our results indicate that, in spite of multiple intermolecular contacts, only the six C-terminal residues of the TRAP cytoplasmic tail bind in an ordered manner to PfAldo. This portion of TRAP targets the PfAldo active site, with its n-1 Trp residue, which is essential for this interaction, buried within the PfAldo catalytic pocket. Docking of a TRAP peptide bearing a Trp to Ala mutation rendered the lower energy configurations either bound weakly outside the active site or not bound to PfAldo at all. The position of the bound TRAP peptide, and particularly the close proximity between the carbonyl of its n-2 Asp residue and the experimentally determined position of the phosphate-6 group of fructose 1,6-phosphate bound to mammalian aldolases, predicts an inhibitory effect of TRAP on catalysis. Enzymatic and TRAP-binding assays using mutant PfAldo molecules strongly support the overall structural model. These results might provide the initial framework for the identification of novel antiparasitic compounds
— id: 70859, year: 2007, vol: 66, page: 528, stat: Journal Article,

Structural basis for coreceptor selectivity by the HIV type 1 V3 loop
Cardozo, Timothy; Kimura, Tetsuya; Philpott, Sean; Weiser, Barbara; Burger, Harold; Zolla-Pazner, Susan
2007 Mar;23(3):415-426, AIDS research & human retroviruses
The third variable region (V3) of the HIV-1 surface glycoprotein, gp120, plays a central role in the interaction of the virus envelope with the cell surface chemokine receptors, triggering membrane fusion and virus entry into human lymphocytes and macrophages. The CXCR4 and CCR5 chemokine receptors are used by 'X4-tropic' and 'R5-tropic' viruses, respectively. Recently, the crown of the V3 loop was shown to bear a close structural homology to the beta2-beta3 loop in the CXC and CC chemokines, the natural ligands of CXCR4 and CCR5, respectively. This homology can serve as the foundation for 3D molecular modeling of the V3 loops from primary isolates whose coreceptor usage was experimentally defined. The modeling revealed a charged 'patch' on the surface of V3 that correlates with coreceptor usage. This V3 surface patch is positively charged in X4-tropic viruses and negatively charged or neutral in R5-tropic viruses, and is formed by two amino acids, at position 11 and at position 24 or 25; amino acids 11 and 24 or 11 and 25 contact each other in 3D space. Residues at positions 11 and 25 were known previously to influence coreceptor usage, and the charge of the residues at these two positions is often used to predict viral tropism. However, we found that the predictive value of using the charge of residues 11, 24, and 25 to identify X4 or R5 tropism was improved over using only the charge of residues 11 and 25. Thus, the data suggest a new ' 11/24/25 rule' : a positively charged amino acid at position 11, 24, or 25 defines X4; otherwise R5. This rule gave an overall predictive value of 94% for 217 viruses whose tropism had been determined experimentally as either X4 or R5. The results have additional implications for the design of HIV therapeutics, vaccines, and strategies for monitoring disease progression
— id: 72077, year: 2007, vol: 23, page: 415, stat: Journal Article,

Wrenches in the works: drug discovery targeting the SCF ubiquitin ligase and APC/C complexes
Cardozo, Timothy; Pagano, Michele
2007 ;8 Suppl 1:S9-S9, BMC biochemistry
Recently, the ubiquitin proteasome system (UPS) has matured as a drug discovery arena, largely on the strength of the proven clinical activity of the proteasome inhibitor Velcade in multiple myeloma. Ubiquitin ligases tag cellular proteins, such as oncogenes and tumor suppressors, with ubiquitin. Once tagged, these proteins are degraded by the proteasome. The specificity of this degradation system for particular substrates lies with the E3 component of the ubiquitin ligase system (ubiquitin is transferred from an E1 enzyme to an E2 enzyme and finally, thanks to an E3 enzyme, directly to a specific substrate). The clinical effectiveness of Velcade (as it theoretically should inhibit the output of all ubiquitin ligases active in the cell simultaneously) suggests that modulating specific ubiquitin ligases could result in an even better therapeutic ratio. At present, the only ubiquitin ligase leads that have been reported inhibit the degradation of p53 by Mdm2, but these have not yet been developed into clinical therapeutics. In this review, we discuss the biological rationale, assays, genomics, proteomics and three-dimensional structures pertaining to key targets within the UPS (SCFSkp2 and APC/C) in order to assess their drug development potential. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)
— id: 75675, year: 2007, vol: 8 Suppl 1, page: S9, stat: Journal Article,

Farnesyl Pyrophosphate is a Novel Transcriptional Activator for a Subset of Nuclear Hormone Receptors
Das, Sharmistha; Schapira, Matthieu; Tomic-Canic, Marjana; Goyanka, Ritu; Cardozo, Timothy; Samuels, Herbert H
2007 Nov;21(11):2672-2686, Molecular endocrinology
In silico docking of a chemical library with the ligand binding domain (LBD) of thyroid hormone nuclear receptor-alpha (TRalpha) suggested that farnesyl pyrophosphate (FPP), a key intermediate in cholesterol synthesis and protein farnesylation, might function as an agonist. Surprisingly, addition of FPP to cells activated TR as well as the classical steroid hormone receptors but not peroxisome proliferative activating receptors, farnesol X receptor, liver x receptor, or several orphan nuclear receptors whose ligands are unknown. FPP enhanced receptor-coactivator binding in vitro and in vivo and elevation of FPP levels in cells by squalene synthetase or farnesyl transferase inhibitors leads to activation. The FPP effect was blocked by selective receptor antagonists, and in silico docking with 143 nuclear receptor LBD structures revealed that FPP only docked with the agonist conformation of those receptors activated by FPP. Our results suggest that certain nuclear receptors maintain a common structural feature that may reflect an action of FPP on an ancient nuclear receptor or that FPP could function as a ligand for one of the many orphan nuclear receptors whose ligands have not yet been identified. This finding also has potential interesting implications which may, in part, explain the pleotropic effects of statins as well as certain actions of farnesylation inhibitors in cells
— id: 73345, year: 2007, vol: 21, page: 2672, stat: Journal Article,

Distinct sequence patterns characterize the V3 region of HIV type 1 gp120 from subtypes A and C
Felsovalyi, Klara; Nadas, Arthur; Zolla-Pazner, Susan; Cardozo, Timothy
2006 Jul;22(7):703-708, AIDS research & human retroviruses
The known sequences of HIV-1 viruses have been categorized into subtypes based on the phylogenetic partitioning of their env and gag gene sequences. The env gene encodes the protein gp120, which contains five sequence- variable regions (V1 to V5), of which the V3 loop is of central importance to viral infectivity. The V3 loop consensus sequences of HIV-1 subtype A and C viruses are similar, and more similar to one another than the V3 consensus sequences of any other two HIV-1 subtypes. However, using a position-specific statistical comparison, we found that the V3 region of these two subtypes is statistically distinct (p = approximately 0.0). (The p-value calculated to the lowest limit of representation on the computer used to run the calculation. This lowest limit was 10(16). Although theoretically a p-value cannot be equal to 0.0, the p-value for the comparisons in question can be intuitively considered to be extremely small, or approximately 0.0.)
— id: 67537, year: 2006, vol: 22, page: 703, stat: Journal Article,

Druggability of SCF ubiquitin ligase-protein interfaces
Cardozo, Timothy; Abagyan, Ruben
2005 ;399:634-653, Methods in enzymology
The unique mechanism of the SCF ubiquitin ligase poses a challenge to drug discovery. A central enzymatic small molecule-binding active site is not evident in this multisubunit protein enzyme, as is the case with kinases or proteases. Instead, the SCF ligase seems to accomplish ubiquitylation through a series of cooperative movements dependent on the protein interfaces between its components and its substrate. Activity-modulating small molecules, therefore, need to interact with these protein interfaces. The three-dimensional structure of these interfaces may be the key asset in determining their suitability for small molecule binding. Computational tools and a systematic approach described in detail here can assess the 'druggability' of an SCF ligase before the investment of effort in high-throughput screening (HTS), structure-based drug design (SBBD), or virtual library screening (VLS)
— id: 78752, year: 2005, vol: 399, page: 634, stat: Journal Article,

Androgen receptor mutations identified in prostate cancer and androgen insensitivity syndrome display aberrant ART-27 coactivator function
Li, Wenhui; Cavasotto, Claudio N; Cardozo, Timothy; Ha, Susan; Dang, Thoa; Taneja, Samir S; Logan, Susan K; Garabedian, Michael J
2005 May 26;19(9):2273-2282, Molecular endocrinology
The transcriptional activity of the androgen receptor (AR) is modulated by interactions with coregulatory molecules. It has been proposed that aberrant interactions between AR and its coregulators may contribute to diseases related to AR activity, such as prostate cancer and androgen insensitivity syndrome (AIS); however, evidence linking abnormal receptor:cofactor interactions to disease is scant. The Androgen Receptor Trapped clone-27 (ART-27) is a recently identified AR N-terminal coactivator that is associated with AR-mediated growth inhibition. Here we analyze a number of naturally occurring AR mutations identified in prostate cancer and AIS for their ability to affect AR response to ART-27. Although the vast majority of AR mutations appeared capable of increased activation in response to ART-27, an AR mutation identified in prostate cancer (AR P340L) and AIS (AR E2K) show reduced transcriptional responses to ART-27, whereas their response to the p160 class of coactivators was not diminished. Relative to the wild-type receptor, less ART-27 protein associated with the AR E2K substitution, consistent with reduced transcriptional response. Surprisingly, more ART-27 associated with AR P340L, despite the fact that the mutation decreased transcriptional activation in response to ART-27. Our findings suggest that aberrant AR-coactivator association interferes with normal ART-27 coactivator function resulting in suppression of AR activity and may contribute to the pathogenesis of diseases related to alterations in AR activity, such as prostate cancer and AIS
— id: 56038, year: 2005, vol: 19, page: 2273, stat: Journal Article,

The SCF ubiquitin ligase: insights into a molecular machine
Cardozo, Timothy; Pagano, Michele
2004 Sep;5(9):739-751, Nature reviews. Molecular cell biology
Ubiquitin ligases are well suited to regulate molecular networks that operate on a post-translational timescale. The F-box family of proteins - which are the substrate-recognition components of the Skp1-Cul1-F-box-protein (SCF) ubiquitin ligase - are important players in many mammalian functions. Here we explore a unifying and structurally detailed view of SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies
— id: 45024, year: 2004, vol: 5, page: 739, stat: Journal Article,

Systematic analysis and nomenclature of mammalian F-box proteins
Jin, Jianping; Cardozo, Timothy; Lovering, Ruth C; Elledge, Stephen J; Pagano, Michele; Harper, J Wade
2004 Nov 1;18(21):2573-2580, Genes & development
— id: 64222, year: 2004, vol: 18, page: 2573, stat: Journal Article,

Eosinophilic fasciitis
Cardozo, Timothy J
2003 Oct;9(4):33-33, Dermatology online journal
The case of a 76-year-old woman with eosinophilic fasciitis is presented. Reported etiologic associations and treatment options are discussed
— id: 45366, year: 2003, vol: 9, page: 33, stat: Journal Article,

Estimating local backbone structural deviation in homology models
Cardozo T; Batalov S; Abagyan R
2000 Jan;24(1):13-31, Computers & chemistry
After the atomic coordinates themselves, the most important data in a homology model are the spatial reliability estimates associated with each of the atoms (atom annotation). Recent blind homology modeling predictions have demonstrated that principally correct sequence-structure alignments are achievable to sequence identities as low as 25% [Martin, A.C., MacArthur, M.W., Thornton, J.M., 1997. Assessment of comparative modeling in CASP2. Proteins Suppl(1), 14-28]. The locations and extent of spatial deviations in the backbone between correctly aligned homologous protein structures remained very poorly estimated however, and these errors were the cause of errant loop predictions [Abagyan, R., Batalov, S., Cardozo, T., Totrov, M., Webber, J., Zhou, Y., 1997. Homology modeling with internal coordinate mechanics: deformation zone mapping and improvements of models via conformational search. Proteins Suppl(1), 29-37]. In order to derive accurate measures for local backbone deviations, we made a systematic study of static local backbone deviations between homologous pairs of protein structures. We found that 'through space' proximity to gaps and chain termini, local three-dimensional 'density', three-dimensional environment conservation, and B-factor of the template contribute to local deviations in the backbone in addition to local sequence identity. Based on these finding, we have identified the meaningful ranges of values within which each of these parameters correlates with static local backbone deviation and produced a combined scoring function to greatly improve the estimation of local backbone deviations. The optimized function has more than twice the accuracy of local sequence identity or B-factor alone and was validated in a recent blind structure prediction experiment. This method may be used to evaluate the utility of a preliminary homology model for a particular biological investigation (e.g. drug design) or to provide an improved starting point for molecular mechanics loop prediction methods
— id: 11859, year: 2000, vol: 24, page: 13, stat: Journal Article,

Genetic linkage mapping of zebrafish genes and ESTs
Kelly, PD; Chu, F; Woods, IG; Ngo-Hazelett, P; Cardozo, T; Huang, H; Kimm, F; Liao, LY; Yan, YL; Zhou, YY; Johnson, SL; Abagyan, R; Schier, AF; Postlethwait, JH; Talbot, WS
2000 APR ;10(4):558-567, Genome research
Generic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes essential for vertebrate development, physiology, and behavior. We have constructed a genetic linkage map that will facilitate the identification of candidate genes for these mutations and allow comparisons among the genomes of zebrafish and other vertebrates. On this map, we have localized 771 zebrafish genes and expressed sequence tags (ESTs) by scoring single-stranded conformational polymorphisms (SSCPs) in a meiotic mapping panel. Of these sequences, 642 represent previously unmapped genes and ESTs. The mapping panel was comprised of 42 homozygous diploid individuals produced by hear shock treatment of haploid embryos at the one-cell stage (HS diploids). This 'doubled haploid' strategy combines the advantages of mapping in haploid and standard diploid systems, because heat shock diploid individuals have only one allele at each locus and can survive to adulthood, enabling a relatively large quantity of genomic DNA to be prepared from each individual in the mapping panel. To integrate this map with others, we also scored 593 previously mapped simple-sequence length polymorphisms (SSLPs) in the mapping panel. This map will accelerate the molecular analysis of zebrafish mutations and facilitate comparative analysis of vertebrate genomes
— id: 54700, year: 2000, vol: 10, page: 558, stat: Journal Article,

A genetic linkage map for zebrafish: comparative analysis and localization of genes and expressed sequences
Gates MA; Kim L; Egan ES; Cardozo T; Sirotkin HI; Dougan ST; Lashkari D; Abagyan R; Schier AF; Talbot WS
1999 Apr;9(4):334-347, Genome research
Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes with essential functions. To facilitate the identification of candidate genes for these mutations, we have genetically mapped 104 genes and expressed sequence tags by scoring single-strand conformational polymorphisms in a panel of haploid siblings. To integrate this map with existing genetic maps, we also scored 275 previously mapped genes, microsatellites, and sequence-tagged sites in the same haploid panel. Systematic phylogenetic analysis defined likely mammalian orthologs of mapped zebrafish genes, and comparison of map positions in zebrafish and mammals identified significant conservation of synteny. This comparative analysis also identified pairs of zebrafish genes that appear to be orthologous to single mammalian genes, suggesting that these genes arose in a genome duplication that occurred in the teleost lineage after the divergence of fish and mammal ancestors. This comparative map analysis will be useful in predicting the locations of zebrafish genes from mammalian gene maps and in understanding the evolution of the vertebrate genome
— id: 56422, year: 1999, vol: 9, page: 334, stat: Journal Article,

The selC-associated SHI-2 pathogenicity island of Shigella flexneri
Moss JE; Cardozo TJ; Zychlinsky A; Groisman EA
1999 Jul;33(1):74-83, Molecular microbiology
Pathogenicity islands are chromosomal gene clusters, often located adjacent to tRNA genes, that encode virulence factors present in pathogenic organisms but absent or sporadically found in related non-pathogenic species. The selC tRNA locus is the site of integration of different pathogenicity islands in uropathogenic Escherichia coli, enterohaemorrhagic E. coli and Salmonella enterica. We show here that the selC locus of Shigella flexneri, the aetiological agent of bacterial dysentery, also contains a pathogenicity island. This pathogenicity island, designated SHI-2 (Shigella island 2), occupies 23.8 kb downstream of selC and contains genes encoding the aerobactin iron acquisition siderophore system, colicin V immunity and several novel proteins. Remnants of multiple mobile genetic elements are present in SHI-2. SHI-2-hybridizing sequences were detected in all S. flexneri strains tested and parts of the island were also found in other Shigella species. SHI-2 may allow Shigella survival in stressful environments, such as those encountered during infection
— id: 56455, year: 1999, vol: 33, page: 74, stat: Journal Article,

The selC associated pathogenicity island of Shigella flexneri and Shigella sonnei: A mosaic structure encoding aerobactin, colicin V immunity, and numerous mobile genetic elements
Moss, J E; Cardozo, T J; Groisman, E A; Zychlinsky, A
1999 May 30-Jun 3;99:30-30, Abstracts of the ... general meeting of the American Society for Microbiology
— id: 15911, year: 1999, vol: 99, page: 30, stat: Journal Article,

Molecular modeling of the domain shared between CED-4 and its mammalian homologue Apaf-1: A structural relationship to the G-proteins
Cardozo, TJ; Abagyan, R
1998 JAN ;4(2):83-93, Journal of molecular modeling
Apoptosis (programmed cell death, PCD) is a characteristic type of cell death in which a regulated cellular response pathway mediated by cysteine proteases of the caspase family and Bcl-2 family proteins results in ordered and non-inflammatory involution of the cell. The CED-4 protein and its recently identified mammalian homologue Apaf-1 are critical but functionally uncharacterized components of the cell death machinery. We present here a three-dimensional molecular model for the central domain of CED-4, its alternatively spliced transcript (CED-41) and Apaf-1. A novel protein family is identified and structure prediction for the family identifies a G-protein-like fold with high reliability. The three-dimensional model provides a potential structural explanation for the alternatively spliced variant as well as the known point mutations in CED-4. Regions of the CED-4 and Apaf-1 sequences which may interact with caspases and the Bcl-2 family are proposed. This new information provides a structural molecular framework for the interaction of CED-4-like proteins with the caspases and the Bcl-2 family in the regulation of apoptosis which is analogous to G-protein mediated interactions in well-defined signal transduction pathways
— id: 53553, year: 1998, vol: 4, page: 83, stat: Journal Article,

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast
Isakoff SJ; Cardozo T; Andreev J; Li Z; Ferguson KM; Abagyan R; Lemmon MA; Aronheim A; Skolnik EY
1998 Sep 15;17(18):5374-5387, EMBO journal
Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K
— id: 7615, year: 1998, vol: 17, page: 5374, stat: Journal Article,

Homology modeling with internal coordinate mechanics: deformation zone mapping and improvements of models via conformational search
Abagyan R; Batalov S; Cardozo T; Totrov M; Webber J; Zhou Y
1997 ;Suppl 1:29-37, Proteins
Five models by homology containing insertions and deletions and ranging from 33% to 48% sequence identity to the known homologue, and one high sequence identity (85%) model were built for the CASP2 meeting. For all five low identity targets: (i) our starting models were improved by the Internal Coordinate Mechanics (ICM) energy optimization, (ii) the refined models were consistently better than those built with the automatic SWISS-MODEL program, and (iii) the refined models differed by less than 2% from the best model submitted, as judged by the residue contact area difference (CAD) measure [Abagyan, R.A., Totrov, M.J. Mol. Biol. 268:678-685, 1997]. The CAD measure is proposed for ranking models built by homology instead of global root-mean-square deviation, which is frequently dominated by insignificant yet large contributions from incorrectly predicted fragments or side chains. We demonstrate that the precise identification of regions of local backbone deviation is an independent and crucial step in the homology modeling procedure after alignment, since aligned fragments can strongly deviate from the template at various distances from the alignment gap or even in the ungapped parts of the alignment. We show that a local alignment score can be used as an indicator of such local deviation. While four short loops of the meeting targets were predicted by database search, the best loop 1 target T0028, for which the correct database fragment was not found, was predicted by Internal Coordinate Mechanics global energy optimization at 1.2 A accuracy. A classification scheme for errors in homology modeling is proposed
— id: 57088, year: 1997, vol: Suppl 1, page: 29, stat: Journal Article,

Expression, purification, and preliminary physicochemical characterization of TSG-14, a cytokine-inducible long pentraxin protein
Goodman, AR; Cardozo, T; Abagyan, R; Lee, GW; Wisniewski, HG; Vilcek, J
1997 JAN ;99(1):56-56, Journal of allergy & clinical immunology
— id: 53277, year: 1997, vol: 99, page: 56, stat: Journal Article,

Long pentraxins: an emerging group of proteins with diverse functions
Goodman AR; Cardozo T; Abagyan R; Altmeyer A; Wisniewski HG; Vilcek J
1996 Aug;7(2):191-202, Cytokine & growth factor reviews
The earliest described pentraxins, C reactive protein (CRP) and serum amyloid P component (SAP), are cytokine-inducible acute phase proteins implicated in innate immunity whose concentrations in the blood increase dramatically upon infection or trauma. The highly conserved family of pentraxins was thought to consist solely of approximately 25 kDa proteins. Recently, several distinct larger proteins have been identified in which only the C-terminal halves show characteristic features of the pentraxin family. One of the recently described 'long' pentraxins (TSG-14/PTX3) is inducible by TNF or IL-1 and is produced during the acute phase response. Other newly identified long pentraxins are constitutively expressed proteins associated with sperm-egg fusion (apexin/p50), may function at the neuronal synapse (neuronal pentraxin I, NPI), or may serve yet other, unknown functions (NPII and XL-PXN1). Evidence obtained by molecular modeling and by direct physicochemical analysis suggests that TSG-14 protein retains some characteristic structural features of the pentraxins, including the formation of pentameric complexes
— id: 12559, year: 1996, vol: 7, page: 191, stat: Journal Article,

Homology modeling by the ICM method
Cardozo T; Totrov M; Abagyan R
1995 Nov;23(3):403-414, Proteins
Five models have been built by the ICM method for the Comparative Modeling section of the Meeting on the Critical Assessment of Techniques for Protein Structure Prediction. The targets have homologous proteins with known three-dimensional structure with sequence identity ranging from 25 to 77%. After alignment of the target sequence with the related three-dimensional structure, the modeling procedure consists of two subproblems: side-chain prediction and loop prediction. The ICM method approaches these problems with the following steps: (1) a starting model is created based on the homologous structure with the conserved portion fixed and the nonconserved portion having standard covalent geometry and free torsion angles; (2) the Biased Probability Monte Carlo (BPMC) procedure is applied to search the subspaces of either all the nonconservative side-chain torsion angles or torsion angles in a loop backbone and surrounding side chains. A special algorithm was designed to generate low-energy loop deformations. The BPMC procedure globally optimizes the energy function consisting of ECEPP/3 and solvation energy terms. Comparison of the predictions with the NMR or crystallographic solutions reveals a high proportion of correctly predicted side chains. The loops were not correctly predicted because imprinted distortions of the backbone increased the energy of the near-native conformation and thus made the solution unrecognizable. Interestingly, the energy terms were found to be reliable and the sampling of conformational space sufficient. The implications of this finding for the strategies of future comparative modeling are discussed
— id: 6893, year: 1995, vol: 23, page: 403, stat: Journal Article,

Motility of vinculin-deficient F9 embryonic carcinoma cells analyzed by video, laser confocal, and reflection interference contrast microscopy
Goldmann WH; Schindl M; Cardozo TJ; Ezzell RM
1995 Dec;221(2):311-319, Experimental cell research
We have studied the motility of wild-type F9 and vinculin-deficient (5.51) mouse embryonal carcinoma cells. F9 cells extended filopodia at a rate of 61 ( +/- 18) nm/s over a distance of 3.18 (+/- 0.29) microns. In contrast, 5.51 cells exhibited filopodia which extended at a similar speed of 57 (+/- 17) nm/s but over a longer distance of 5.10 (+/- 2.14) microns. Cell-substratum contact areas of both cell types were examined by reflection interference contrast microscopy. Wild-type F9 cells had distinct close contacts (dark gray areas) at the cell periphery, whereas 5.51 cells had only a few light gray pinpoint contacts with the substrate. Confocal microscopy showed alpha-actinin to be localized along actin stress fibers in wild-type cells, and in 5.51 cells stress fibers were absent and alpha-actinin was associated with F-actin in the filopodia. beta 1-integrin, talin, and paxillin were concentrated in focal contacts in wild-type cells, but in 5.51 cells beta 1-integrin and talin were in patches under the plasma membrane and paxillin was diffusely distributed in the cytoplasm. We conclude that changes in cell shape and motility of 5.51 compared to wild-type F9 cells are due to the absence of vinculin even though there may be functions of other focal adhesion complex proteins, e.g., talin, linking the actin cytoskeleton to the plasma membrane
— id: 45367, year: 1995, vol: 221, page: 311, stat: Journal Article,

Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell
Samuels M; Ezzell RM; Cardozo TJ; Critchley DR; Coll JL; Adamson ED
1993 May;121(4):909-921, Journal of cell biology
A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell-substrate adhesion. We have previously shown that neither uvomorulin (E-cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F-actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins
— id: 45368, year: 1993, vol: 121, page: 909, stat: Journal Article,

Expression and localization of villin, fimbrin, and myosin I in differentiating mouse F9 teratocarcinoma cells
Ezzell RM; Leung J; Collins K; Chafel MM; Cardozo TJ; Matsudaira PT
1992 Jun;151(2):575-585, Developmental biology (Orlando)
F9 embryonic carcinoma cells are a multipotent cell line which can be induced to differentiate into cells resembling the visceral endoderm, an extraembryonic absorptive epithelium characterized by apical microvilli. We have examined the role of villin, fimbrin, and myosin I, the major actin-binding proteins in the intestinal and visceral yolk sac microvilli, in the development of epithelial polarity and the assembly of the microvillus cytoskeleton in differentiating F9 cells. By immunoblot analysis villin was first detected at 4 days of differentiation. Confocal microscopy localized villin at Day 4 to the apical surface and by Day 6 to the basolateral surfaces as well. In comparison, fimbrin and myosin I were both present in undifferentiated F9 cells and became associated with the apical surface after villin during differentiation to visceral endoderm. The accumulation of villin, fimbrin, and myosin I at the apical surface in differentiating F9 cells correlated with the appearance of microvilli containing organized actin filament bundles. Two mouse villin cDNAs were isolated and characterized to examine villin expression during F9 differentiation. Mouse villin was encoded by two transcripts (3.8 and 3.4 kb) which differ in their 3'-noncoding region. Both villin mRNAs were first detected by Day 4 of differentiation and their appearance coincided with expression of the visceral endoderm marker alpha-fetoprotein. The pattern of expression and order of accumulation of villin, fimbrin, and myosin I in differentiating F9 cells are common to developing gut and yolk sac epithelium. This suggests that microvillus assembly is directed by a sequence of temporally and spatially regulated localizations of these actin-binding proteins
— id: 45369, year: 1992, vol: 151, page: 575, stat: Journal Article,