RESUMO
Antibodies to DNA and chromatin drive autoimmunity in systemic lupus erythematosus (SLE). Null mutations and hypomorphic variants of the secreted deoxyribonuclease DNASE1L3 are linked to familial and sporadic SLE, respectively. We report that DNASE1L3-deficient mice rapidly develop autoantibodies to DNA and chromatin, followed by an SLE-like disease. Circulating DNASE1L3 is produced by dendritic cells and macrophages, and its levels inversely correlate with anti-DNA antibody response. DNASE1L3 is uniquely capable of digesting chromatin in microparticles released from apoptotic cells. Accordingly, DNASE1L3-deficient mice and human patients have elevated DNA levels in plasma, particularly in circulating microparticles. Murine and human autoantibody clones and serum antibodies from human SLE patients bind to DNASE1L3-sensitive chromatin on the surface of microparticles. Thus, extracellular microparticle-associated chromatin is a potential self-antigen normally digested by circulating DNASE1L3. The loss of this tolerance mechanism can contribute to SLE, and its restoration may represent a therapeutic opportunity in the disease.
Assuntos
Autoanticorpos/imunologia , Micropartículas Derivadas de Células/química , Cromatina/imunologia , DNA/imunologia , Endodesoxirribonucleases/genética , Lúpus Eritematoso Sistêmico/imunologia , Animais , Micropartículas Derivadas de Células/metabolismo , Modelos Animais de Doenças , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Humanos , Células Jurkat , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.
Assuntos
Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cisteína/metabolismo , Células HEK293 , Humanos , Canais KATP/genética , Lipoilação/fisiologia , Mutagênese/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp/métodos , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Cultura Primária de Células , Ratos , Receptores de Sulfonilureias/genéticaRESUMO
BACKGROUND: Emerging resistance of the malaria parasite Plasmodium to current therapies underscores the critical importance of exploring novel strategies for disease eradication. Plasmodium species are obligate intracellular protozoan parasites. They rely on an unusual form of substrate-dependent motility for their migration on and across host-cell membranes and for host cell invasion. This peculiar motility mechanism is driven by the 'glideosome', an actin-myosin associated, macromolecular complex anchored to the inner membrane complex of the parasite. Myosin A, actin, aldolase, and thrombospondin-related anonymous protein (TRAP) constitute the molecular core of the glideosome in the sporozoite, the mosquito stage that brings the infection into mammals. METHODS: Virtual library screening of a large compound library against the PfAldolase-TRAP complex was used to identify candidate compounds that stabilize and prevent the disassembly of the glideosome. The mechanism of these compounds was confirmed by biochemical, biophysical and parasitological methods. RESULTS: A novel inhibitory effect on the parasite was achieved by stabilizing a protein-protein interaction within the glideosome components. Compound 24 disrupts the gliding and invasive capabilities of Plasmodium parasites in in vitro parasite assays. A high-resolution, ternary X-ray crystal structure of PfAldolase-TRAP in complex with compound 24 confirms the mode of interaction and serves as a platform for future ligand optimization. CONCLUSION: This proof-of-concept study presents a novel approach to anti-malarial drug discovery and design. By strengthening a protein-protein interaction within the parasite, an avenue towards inhibiting a previously "undruggable" target is revealed and the motility motor responsible for successful invasion of host cells is rendered inactive. This study provides new insights into the malaria parasite cell invasion machinery and convincingly demonstrates that liver cell invasion is dramatically reduced by 95 % in the presence of the small molecule stabilizer compound 24.
Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/química , Proteínas de Protozoários/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Frutose-Bifosfato Aldolase/química , Hepatócitos/efeitos dos fármacos , Humanos , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Complexos Multiproteicos/efeitos dos fármacos , Plasmodium falciparum/química , Estabilidade Proteica/efeitos dos fármacos , Proteínas de Protozoários/química , Coelhos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/toxicidade , Ressonância de Plasmônio de SuperfícieRESUMO
Metabolism is a fundamental cellular process that is coordinated with cell cycle progression. Despite this association, a mechanistic understanding of cell cycle phase-dependent metabolic pathway regulation remains elusive. Here we report the mechanism by which human de novo pyrimidine biosynthesis is allosterically regulated during the cell cycle. Combining traditional synchronization methods and metabolomics, we characterize metabolites by their accumulation pattern during cell cycle phases and identify cell cycle phase-dependent regulation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase (CAD), the first, rate-limiting enzyme in de novo pyrimidine biosynthesis. Through systematic mutational scanning and structural modelling, we find allostery as a major regulatory mechanism that controls the activity change of CAD during the cell cycle. Specifically, we report evidence of two Animalia-specific loops in the CAD allosteric domain that involve sensing and binding of uridine 5'-triphosphate, a CAD allosteric inhibitor. Based on homology with a mitochondrial carbamoyl-phosphate synthetase homologue, we identify a critical role for a signal transmission loop in regulating the formation of a substrate channel, thereby controlling CAD activity.
Assuntos
Carbamoil Fosfato Sintase (Glutamina-Hidrolizante) , Pirimidinas , Humanos , Regulação Alostérica , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/química , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Ciclo Celular , Pirimidinas/farmacologia , FosfatosRESUMO
Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked "hits", which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9 µM and 11 µM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.
RESUMO
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/.
Assuntos
Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas , Proteômica/métodos , Algoritmos , Animais , Simulação por Computador , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Estatísticos , Conformação Molecular , Ligação Proteica , Proteínas Quinases/química , Proteína Fosfatase 2/química , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , SoftwareRESUMO
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.
Assuntos
Drosophila/metabolismo , Transativadores/genética , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Drosophila/genética , Fosforilação , Proteínas/genética , Proteínas/metabolismo , Transativadores/metabolismoRESUMO
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.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Neurônios/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/isolamento & purificação , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Voltage-gated ion channels (VGICs) are associated with hundreds of human diseases. To date, 3D structural models of human VGICs have not been reported. We developed a 3D structural integrity metric to rank the accuracy of all VGIC structures deposited in the PDB. The metric revealed inaccuracies in structural models built from recent single-particle, non-crystalline cryo-electron microscopy maps and enabled the building of highly accurate homology models of human Cav channel α1 subunits at atomic resolution. Human Cav Mendelian mutations mostly located to segments involved in the mechanism of voltage sensing and gating within the 3D structure, with multiple mutations targeting equivalent 3D structural locations despite eliciting distinct clinical phenotypes. The models also revealed that the architecture of the ion selectivity filter is highly conserved from bacteria to humans and between sodium and calcium VGICs.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Modelos Moleculares , Mutação , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Domínios ProteicosRESUMO
Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Receptores de Esteroides/fisiologia , Transcrição Gênica/efeitos dos fármacos , Animais , Linhagem Celular , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Camundongos , Receptor de Pregnano X , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Esteroides/metabolismo , Receptores X de Retinoides/metabolismoRESUMO
BACKGROUND: Only 2% of mothers positive for anti-SSA/Ro (Ro) antibodies have children with congenital heart block (CHB). This study aimed to determine whether reactivity with p305, an epitope within the α1G T-type calcium channel, confers added risk over anti-Ro antibodies. METHODS AND RESULTS: Using sera from anti-Ro-exposed pregnancies resulting in offspring with CHB, no disease but CHB-sibling, and no disease and no CHB-sibling, as well as disease (lupus without anti-Ro) and healthy controls, reactivities were determined for binding to Ro60, p305, and an epitope within Ro60, p133-Ro60, which shares structural properties with p305, including key amino acids and an α-helical structure. Candidate peptides were further evaluated in an in vitro model that assessed the binding of maternal antibodies to apoptotic cells. In anti-Ro-positive mothers, anti-p305 autoantibodies (>3 SD above healthy controls) were detected in 3/59 (5%) CHB pregnancies, 4/30 (13%) unaffected pregnancies with a CHB-sibling, and 0/42 (0%) of unaffected pregnancies with no CHB-sibling. For umbilical bloods (61 CHB, 41 healthy with CHB sibling), no association of anti-p305 with outcome was detected; however, overall levels of anti-p305 were elevated compared to mothers during pregnancy in all groups studied. For anti-p133-Ro60, reactivity paralleled that of anti-p305. In the screen employing apoptotic cells, p133-Ro60, but not p305, significantly attenuated the binding of immunoglobulin G isolated from a mother whose child had CHB (42.1% reduced to 13.9%, absence/presence of p133-Ro60, respectively, P<0.05). CONCLUSIONS: These data suggest that anti-p305 is not a robust maternal marker for assessing increased risk of CHB during an anti-SSA/Ro pregnancy.
Assuntos
Anticorpos Antinucleares/imunologia , Doenças Autoimunes/complicações , Canais de Cálcio Tipo T/imunologia , Bloqueio Cardíaco/congênito , Adulto , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/genética , Apoptose , Autoanticorpos/sangue , Autoanticorpos/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Biomarcadores/sangue , Canais de Cálcio Tipo T/sangue , Canais de Cálcio Tipo T/genética , Epitopos/sangue , Epitopos/imunologia , Feminino , Bloqueio Cardíaco/sangue , Bloqueio Cardíaco/imunologia , Humanos , Recém-Nascido , Gravidez , Fatores de Risco , Adulto JovemRESUMO
The case of a 76-year-old woman with eosinophilic fasciitis is presented. Reported etiologic associations and treatment options are discussed.
Assuntos
Eosinofilia , Fasciite , Idoso , Eosinofilia/tratamento farmacológico , Eosinofilia/etiologia , Eosinofilia/patologia , Fasciite/tratamento farmacológico , Fasciite/etiologia , Fasciite/patologia , Feminino , Humanos , Imunossupressores/uso terapêuticoRESUMO
The ranking of scores of individual chemicals within a large screening library is a crucial step in virtual screening (VS) for drug discovery. Previous studies showed that the quality of protein-ligand recognition can be improved using spectrum properties and the shape of the binding energy landscape. Here, we investigate whether the energy gap, defined as the difference between the lowest energy pose generated by a docking experiment and the average energy of all other generated poses and inferred to be a measure of the binding energy landscape sharpness, can improve the separation power between true binders and decoys with respect to the use of the best docking score. We performed retrospective single- and multiple-receptor conformation VS experiments in a diverse benchmark of 40 domains from 38 therapeutically relevant protein targets. Also, we tested the performance of the energy gap on 36 protein targets from the Directory of Useful Decoys (DUD). The results indicate that the energy gap outperforms the best docking score in its ability to discriminate between true binders and decoys, and true binders tend to have larger energy gaps than decoys. Furthermore, we used the energy gap as a descriptor to measure the height of the native binding phase and obtained a significant increase in the success rate of near native binding pose identification when the ligand binding conformations within the boundaries of the native binding phase were considered. The performance of the energy gap was also evaluated on an independent test case of VS-identified PKR-like ER-localized eIF2α kinase (PERK) inhibitors. We found that the energy gap was superior to the best docking score in its ability to more highly rank active compounds from inactive ones. These results suggest that the energy gap of the protein-ligand binding energy landscape is a valuable descriptor for use in VS.
Assuntos
Proteínas/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas/químicaRESUMO
In the ubiquitin proteasome system, the E3 ligase SCF-Skp2 and its accessory protein, Cks1, promote proliferation largely by inducing the degradation of the CDK inhibitor p27. Overexpression of Skp2 in human cancers correlates with poor prognosis, and deregulation of SCF-Skp2-Cks1 promotes tumorigenesis in animal models. We identified small molecule inhibitors specific to SCF-Skp2 activity using in silico screens targeted to the binding interface for p27. These compounds selectively inhibited Skp2-mediated p27 degradation by reducing p27 binding through key compound-receptor contacts. In cancer cells, the compounds induced p27 accumulation in a Skp2-dependent manner and promoted cell-type-specific blocks in the G1 or G2/M phases. Designing SCF-Skp2-specific inhibitors may be a novel strategy to treat cancers dependent on the Skp2-p27 axis.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Ubiquitinação/efeitos dos fármacosRESUMO
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200-3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100-200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169-184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100-800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100-3200) and 3570 (range: 200-12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.