Doron Gerber
Bar-Ilan University
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Featured researches published by Doron Gerber.
Nature Biotechnology | 2008
Shirit Einav; Doron Gerber; Paul D. Bryson; Ella H. Sklan; Menashe Elazar; Sebastian J. Maerkl; Jeffrey S. Glenn; Stephen R. Quake
More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3′ terminus of the negative strand of the viral genome with a dissociation constant (Kd) of ∼3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4Bs RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.
Nature Biotechnology | 2010
Polly M. Fordyce; Doron Gerber; Danh Tran; Jiashun Zheng; Hao Li; Joseph L. DeRisi; Stephen R. Quake
Gene expression is regulated in part by protein transcription factors that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link transcription factors to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 Saccharomyces cerevisiae transcription factors with a variety of DNA-binding domains, including several that have proven difficult to study by other techniques. For each transcription factor, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for four transcription factors, we also determined absolute affinities. We expect that these data and future use of this technique will provide information essential for understanding transcription factor specificity, improving identification of regulatory sites and reconstructing regulatory interactions.
Nature Methods | 2009
Doron Gerber; Sebastian J. Maerkl; Stephen R. Quake
We developed an in vitro protein expression and interaction analysis platform based on a highly parallel and sensitive microfluidic affinity assay, and used it for 14,792 on-chip experiments, which exhaustively measured the protein-protein interactions of 43 Streptococcus pneumoniae proteins in quadruplicate. The resulting network of 157 interactions was denser than expected based on known networks. Analysis of the network revealed previously undescribed physical interactions among members of some biochemical pathways.
Journal of Biological Chemistry | 2007
Neta Sal-Man; Doron Gerber; Itai Bloch; Yechiel Shai
Aromatic residues have been previously shown to mediate the self-assembly of different soluble proteins through π-π interactions (McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458–15463). However, their role in transmembrane (TM) assembly is not yet clear. In this study, we performed statistical analysis of the frequency of occurrence of aromatic pairs in a bacterial TM data base that provided an initial indication that the appearance of a specific aromatic pattern, Aromatic-XX-Aromatic, is not coincidental, similar to the well characterized QXXS motif. The QXXS motif was previously shown to be both critical and sufficient for stabilizing TM self-assembly. Using the ToxR system, we monitored the dimerization propensities of TM domains that contain mutations of interacting residues to aromatic amino acids and demonstrated that aromatic residues can adequately stabilize self-association. Importantly, we have provided an example of a natural TM domain, the cholera toxin secretion protein EpsM, whose TM self-assembly is mediated by an aromatic motif (WXXW). This is, in fact, the first evidence that aromatic residues are involved in the dimerization of a wild type TM domain. The association mediated by aromatic residues was found to be sensitive to the TM sequence, suggesting that aromatic residue motifs can provide a general means for specificity in TM assembly. Molecular dynamics provided a structural explanation for this backbone sequence sensitivity.
Biochimica et Biophysica Acta | 2012
Avner Fink; Neta Sal-Man; Doron Gerber; Yechiel Shai
Protein-protein interactions within the membrane are involved in many vital cellular processes. Consequently, deficient oligomerization is associated with known diseases. The interactions can be partially or fully mediated by transmembrane domains (TMD). However, in contrast to soluble regions, our knowledge of the factors that control oligomerization and recognition between the membrane-embedded domains is very limited. Due to the unique chemical and physical properties of the membrane environment, rules that apply to interactions between soluble segments are not necessarily valid within the membrane. This review summarizes our knowledge on the sequences mediating TMD-TMD interactions which include conserved motifs such as the GxxxG, QxxS, glycine and leucine zippers, and others. The review discusses the specific role of polar, charged and aromatic amino acids in the interface of the interacting TMD helices. Strategies to determine the strength, dynamics and specificities of these interactions by experimental (ToxR, TOXCAT, GALLEX and FRET) or various computational approaches (molecular dynamic simulation and bioinformatics) are summarized. Importantly, the contribution of the membrane environment to the TMD-TMD interaction is also presented. Studies utilizing exogenously added TMD peptides have been shown to influence in vivo the dimerization of intact membrane proteins involved in various diseases. The chirality independent TMD-TMD interactions allows for the design of novel short d- and l-amino acids containing TMD peptides with advanced properties. Overall these studies shed light on the role of specific amino acids in mediating the assembly of the TMDs within the membrane environment and their contribution to protein function. This article is part of a Special Issue entitled: Protein Folding in Membranes.
Journal of Biological Chemistry | 2000
Doron Gerber; Yechiel Shai
The pore-forming domain of Bacillus thuringiensis Cry1Ac insecticidal protein comprises of a seven α-helix bundle (α1–α7). According to the “umbrella model,” α4 and α5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, α4-loop-α5, its α4 and α5 helices, as well as mutant α4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the α4-loop-α5 hairpin is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of α4-loop-α5 as the membrane-inserted pore-forming hairpin in which α4 and α5 line the lumen of the channel and α5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active α4 mutant peptide completely inhibits α4-loop-α5 pore formation, thus providing, to our knowledge, the first example that a mutated helix within a pore can function as an “immunity protein” by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and function of other membrane proteins as well.
PLOS Pathogens | 2012
Gregory Neveu; Rina Barouch-Bentov; Amotz Zivav; Doron Gerber; Yves Jacob; Shirit Einav
Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that cores YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development.
Journal of Clinical Investigation | 2005
Francisco J. Quintana; Doron Gerber; Sally C. Kent; Irun R. Cohen; Yechiel Shai
The fusion peptide (FP) in the N terminus of the HIV envelope glycoprotein, gp41, functions together with other gp41 domains to fuse the virion with the host cell membrane. We now report that FP colocalizes with CD4 and TCR molecules, coprecipitates with the TCR, and inhibits antigen-specific T cell proliferation and proinflammatory cytokine secretion in vitro. These effects are specific: T cell activation by PMA/ionomycin or mitogenic antibodies is not affected by FPs, and FPs do not interfere with antigen-presenting cell function. In vivo, FPs inhibit the activation of arthritogenic T cells in the autoimmune disease model of adjuvant arthritis and reduce the disease-associated IFN-gamma response. Hence, FPs might play 2 roles in HIV infection: mediating membrane fusion while downregulating T cell responses to itself that could block infection. Disassociated from HIV, however, the FP molecule provides a novel reagent for downregulating undesirable immune responses, exemplified here by adjuvant arthritis.
The FASEB Journal | 2005
Doron Gerber; Francisco J. Quintana; Itai Bloch; Irun R. Cohen; Yechiel Shai
T cell activation requires the cross‐talk between the CD3‐signaling complex and the T cell receptor (TCR). A synthetic peptide coding for the TCRa transmembrane domain (CP) binds CD3 molecules, interferes with the CD3/TCR cross‐talk, and inhibits T cell activation. Intermolecular interactions are sterically constrained; accordingly no sequence‐specific interactions are thought to occur between D‐ and L‐stereoisomers. This argument was recently challenged when applied to intra‐membrane protein assembly. In this paper we studied the ability of a D‐stereoisomer of CP (D‐CP) to inhibit T cell activation. L‐CP and D‐CP co‐localized with the TCR in the membrane and inhibited T cell activation in a sequence‐specific manner. In vivo, both L‐CP and D‐CP inhibited adjuvant arthritis. In molecular terms, these results suggest the occurrence of structural reorientation that facilitates native‐like interactions between D‐CP and CD3 within the membrane. In clinical terms, our results demonstrate that D‐stereoisomers retain the therapeutic properties of their L‐stereoisomers, while they benefit from an increased resistance to degradation.
The FASEB Journal | 2007
Itai Bloch; Francisco J. Quintana; Doron Gerber; Tomer Cohen; Irun R. Cohen; Yechiel Shai
Fusion peptide (FP) of the HIV gp41 molecule inserts into the T cell membrane during virus‐cell fusion. FP also blocks the TCR/CD3 interaction needed for antigen‐triggered T cell activation. Here we used in vitro (fluorescence and immunoprecipitation), in vivo (T cell mediated autoimmune disease adjuvant arthritis), and in silico methods to identify the FP‐TCR novel interaction motif: the α‐helical transmembrane domain (TMD) of the TCR α chain, and the β‐sheet 5–13 region of the 16 N‐terminal aa of FP (FP1–16). Deciphering the molecular mechanism of the immunosuppressive activity of FP provides a new potential target to overcome the immunosuppressant activity of HIV, and in addition a tool for down‐regulating immune mediated inflammation.—Bloch, I., Quintana, F. J., Gerger, D., Cohen, T., Cohen, I. R., and Shai, Y. T‐Cell inactivation and immunosuppressive activity induced by HIV gp41 via novel interacting motif. FASEB J. 21, 393–401 (2007)