Yossef Kliger - Academia.edu (original) (raw)

Papers by Yossef Kliger

Frontiers in Immunology, Dec 11, 2017

Background: The expression of heat shock protein gp96 is strongly correlated with the degree of t... more Background: The expression of heat shock protein gp96 is strongly correlated with the degree of tissue inflammation in ulcerative colitis and Crohn's disease, thereby leading us to the hypothesis that inhibition of expression via gp96-II peptide prevents intestinal inflammation.

Journal of Molecular Biology, Aug 1, 2000

The human immunode®ciency virus type 1 gp41 ectodomain forms a three-hairpin protease-resistant c... more The human immunode®ciency virus type 1 gp41 ectodomain forms a three-hairpin protease-resistant core in the absence of membranes, namely, the putative gp41 fusion-active state. Here, we show that recombinant proteins corresponding to the ectodomain of gp41, but lacking the fusion peptide, bind membranes and consequently undergo a major conformational change. As a result, the protease-resistant core becomes susceptible to proteolytic digestion. Accordingly, synthetic peptides corresponding to the segments that construct this core bind the membrane. It is remarkable that the hetero-oligomer formed by these peptides dissociates upon binding to the membrane. These results are consistent with a model in which, after the three-hairpin conformation is formed, membrane binding induces opening of the gp41 core complex. We speculate that binding of the segments that constructed the core to the viral and cellular membranes could bring the membranes closer together and facilitate their merging.

Protein Engineering Design & Selection, Jan 12, 2010

Edited by Amnon Horovitz Correlated mutation analysis (CMA) is a sequence-based approach for ab i... more Edited by Amnon Horovitz Correlated mutation analysis (CMA) is a sequence-based approach for ab initio protein contact map prediction. The basis of this approach is the observed correlation between mutations in interacting amino acid residues. These correlations are often estimated by either calculating the Pearson's correlation coefficient (PCC) or the mutual information (MI) between columns in a multiple sequence alignment (MSA) of the protein of interest and its homologs. A major challenge of CMA is to filter out the background noise originating from phylogenetic relatedness between sequences included in the MSA. Recently, a procedure to reduce this background noise was demonstrated to improve an MI-based predictor. Herein, we tested whether a similar approach can also improve the performance of the classical PCC-based method. Indeed, performance improvements were achieved for all four major SCOP classes. Furthermore, the results reveal that the improved PCC-based method is superior to MI-based methods for proteins having MSAs of up to 100 sequences.

Drug Discovery Today, Mar 1, 2005

The severe acute respiratory syndrome (SARS) epidemic brought into the spotlight the need for rap... more The severe acute respiratory syndrome (SARS) epidemic brought into the spotlight the need for rapid development of effective anti-viral drugs against newly emerging viruses. Researchers have leveraged the 20-year battle against AIDS into a variety of possible treatments for SARS. Most prominently, based solely on viral genome information, silencers of viral genes, viral-enzyme blockers and viral-entry inhibitors were suggested as potential therapeutic agents for SARS. In particular, inhibitors of viral entry, comprising therapeutic peptides, were based on the recently launched anti-HIV drug enfuvirtide. This could represent one of the most direct routes from genome sequencing to the discovery of antiviral drugs.

Journal of Molecular Biology, 1999

Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently... more Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently to inhibit virus-cell fusion. To ®nd out their possible role in the fusion process, two biologically active heptad-repeat segments of the fusion protein (F) of Sendai virus, SV-150 (residues 150-186), and SV-473 (residues 473-495) were synthesized,¯uorescently labeled and spectroscopically characterized for their structure and organization in solution and within the membrane. SV-150 was found to be 50fold less active than SV-473 in inhibiting Sendai virus-cell fusion. Circular dichroism (CD) spectroscopy revealed that in aqueous solution, the peptides are self-associated and adopt low a-helical structure. However, when the two peptides are mixed together, their a-helical content signi®cantly increases. Fluorescence studies, CD, and polarized attenuated total re¯ection infrared (ATR-FTIR) spectroscopy showed that both peptides, alone or as a complex, bind strongly to negatively charged and zwitterionic phospholipid membranes, dissociate therein into a-helical monomers, but do not perturb the lipid packing of the membrane. The ability of the peptides to interact with each other in solution may be correlated with antiviral activity, whereas their ability to interact with the membrane, together with their location near the fusion peptide and the transmembrane domain, suggests a revision to the currently accepted model for viral-induced membrane fusion. In the revised model, in the sequence of events associated with viral entry, the two heptad-repeat sequences may assist in bringing the viral and cellular membranes closer, thus facilitating membrane fusion.

Biochemistry, Apr 1, 1997

HIV-1 transmembrane envelope glycoprotein (gp41) has an unusually long cytoplasmic domain that ha... more HIV-1 transmembrane envelope glycoprotein (gp41) has an unusually long cytoplasmic domain that has secondary associations with the inner leaflet of the membrane. Two highly amphiphatic R-helices in the cytoplasmic domain of gp41 have previously been shown to interact with lipid bilayers. We have detected a highly conserved leucine zipper-like sequence between the two R-helices. A peptide corresponding to this segment (residues 789-815, LLP-3) aggregates in aqueous solution, but spontaneously inserts into phospholipid membranes and dissociates into R-helical monomers. The peptide perturbs the bilayer structure resulting in the formation of micelles and other non-bilayer structures. Tryptophan fluorescence quenching experiments using brominated phospholipids revealed that the peptide penetrates deeply into the hydrophobic milieu of the membrane bilayer. The peptide interacts equally with zwitterionic and negatively-charged phospholipid membranes and is protected from proteolytic digestion in its membrane-bound state. Polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy showed that the LLP-3 R-helix axis is about 70°from the normal to the membrane plane. The ATR-FTIR CH 2-stretching dichroic ratio increases when the peptide is incorporated into pure phospholipid membranes, further indicating that the peptide can deeply penetrate and perturb the bilayer structure. Integrating these data with what is already known about the membrane-associating features of adjacent segments, we propose a revised structural model in which a large portion of the cytoplasmic tail of the HIV-1 envelope glycoprotein is associated with the membrane.

Proceedings of the National Academy of Sciences of the United States of America, Aug 18, 2009

Journal of Biological Chemistry, Dec 1, 2008

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical th... more G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

Journal of Molecular Biology, 2000

HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). Th... more HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and con®rmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial a-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.

Regular and Young Investigator Award Abstracts, Nov 1, 2022

Background Tertiary lymphoid structures (TLS) recently emerged as an intra-tumoral niches of immu... more Background Tertiary lymphoid structures (TLS) recently emerged as an intra-tumoral niches of immune-cell aggregates with a predictive value for cancer immunotherapy responses. 1 LAMP3 + DCs in the TLS were shown to interact and support the differentiation of stem-like CD8 T-cells into effector-like cells, that then expand in the tumor micro-environment (TME) and may exert anti-tumor responses. 2 We investigated the expression of DNAM-1 axis genes: PVRIG, TIGIT, DNAM-1 and their ligands PVRL2 and PVR in the TME. 3 Methods MERFISH technology was employed to detect the expression of 350 distinct mRNA transcripts at sub-cellular resolution in CRC sections. Publicly available TME scRNA-seq datasets were analyzed for expression of PVRIG and PVRL2 across immune populations and validated by flow-cytometry. An extensive omics profiling was performed for patients with pre-and on-treatment biopsies from COM701 (anti-PVRIG antibody) and COM701+nivolumab Phase-1 study (NCT03667716). Results Spatial distribution of gene transcripts allowed identifying localization of stem-like T-cells in TLS regions of two CRC patients (figure 1, p<0.001). While, CTLA-4, PD-1, and TIM3 were mainly expressed by tumor infiltrating T cells, PVRIG and other genes of DNAM-1 axis were also largely expressed in tumor bed, and even more intensely in TLS (p<0.05, figure 2). Furthermore, high resolution unsupervised scRNA gene co-expression analysis in the TME further validated that while PD-1 is strongly correlated with TIM3, CTLA-4, and other markers of exhausted T-cells, PVRIG uniquely clusters with markers of stem-like T-cells. The PVRIG protein expression was increased on CD28+ stem-like T-cells across indications (figure 3). RNA and protein expression data identified PVRL2, PVRIG ligand, preferentially expressed across DC-subtypes compared to PD-L1 and PVR (figure 4). PVRIG blockade could therefore enhance memory T-cell activation by DCs, resulting in their increased expansion and differentiation. Accordingly, COM701 monotherapy induced CD8 + T-cell numbers and immune activation in the TME of ovarian cancer patient (figure 5). Moreover, MSS-CRC patient with partial response to COM701+nivolumab, demonstrated an increase in TCR numbers, clonality, T-cell infiltration and activation in the TME (figure 6). Finally, preliminary analysis of serum from two patients clinically responding to COM701 +nivolumab (RECIST criteria), revealed induction of activated-DC markers, compared to non-responders (figure 7). Conclusions By leveraging spatial and scRNA transcriptomics, we identified PVRIG + CD8 + T-cells predominantly localized within TLS, interacting with PVRL2 + LAMP3 + DCs. PVRIG blockade could therefore enhance the differentiation and expansion of stem-like CD8 + T-cells into effector cells (figure 8). Accordingly, early clinical data shows increased T-cells infiltration and immune activation in patients treated with COM701 or COM701+nivolumab.

Journal for ImmunoTherapy of Cancer, Nov 1, 2021

Background T-cell accumulation in tumors is a prerequisite for response to cancer immunotherapy. ... more Background T-cell accumulation in tumors is a prerequisite for response to cancer immunotherapy. Recent studies highlighted the importance of an early-memory (stem-like) T-cell sub-population, that can self-renew and differentiate into effector cells, and of dendritic cells (DCs), which are essential for T-cell priming and expansion following checkpoint blockade. 1 2 PVRIG is a novel inhibitory receptor that competes with the co-activating receptor DNAM-1, for the binding of a shared ligand, PVRL2. PVRIG expression is induced on T and NK tumor infiltrating cells, whereas PVRL2 is expressed on tumor, endothelial and myeloid cells in the tumor micro-environment (TME). 3 We investigated the expression of PVRIG and PVRL2 across TME immune subpopulations. Methods Publicly available TME scRNA sequencing datasets were analyzed for the expression of PVRIG and PVRL2 across immune subsets. Unsupervised principal-component-analysis and hierarchical co-expression pattern among genes known to be expressed on naïve, memory, and exhausted CD8+ T-cells was performed. Observations were validated by flow-cytometry and immunohistochemistry analysis across a variety of tumor indications. Proximity Extension Assay (PEA, Olink) was conducted using serums collected at several time-points from COM701 (anti-PVRIG antibody) and nivolumab treated patients in a Phase-1 study (NCT03667716). Results Across scRNA datasets, PVRIG, like TIGIT and PD-1, was expressed by both stem-like (TCF1+PD1+) and exhausted (TIM3+CD39+) CD8+ T-cells. High resolution unsupervised scRNA gene co-expression analysis revealed that while TIGIT is strongly correlated with PD-1, CTLA-4, and other markers of exhausted T-cells, PVRIG uniquely clusters with markers of early memory T-cells (figure 1). Accordingly, PVRIG protein expression was increased on CD28+ earlymemory T-cells across indications (figure 2).RNA expression data also revealed that PVRL2 is more abundantly expressed across DC-subtypes compared to PD-L1 and PVR (ligand of TIGIT, figure 3). Flow cytometry confirmed dominant PVRL2 expression on DC subtypes across tumor indications. Immunohistochemistry analysis identified PVRL2 expression in Tertiary Lymphoid Structures (figure 4). Finally, preliminary analysis of serum from COM701+nivolumab treated patients revealed elevated induction of activated-DC markers in two patients that responded clinically (RECIST criteria), compared to nonresponders (figure 5). Conclusions PVRIG is co-expressed with PD-1 and TIGIT on stem-like and exhausted T cells but has a unique dominant expression on early memory cells, while PVRL2 is abundantly expressed across DC-types. PVRIG blockade could therefore enhance memory T-cells activation by DCs, resulting in their increased expansion and differentiation. Accordingly, early data shows increased induction of activated DC markers, potentially following efficient T-DC interaction, in serum of two patients responding to COM701+nivolumab.

PLOS ONE, Dec 27, 2006

Identification of homologous proteins provides a basis for protein annotation. Sequence alignment... more Identification of homologous proteins provides a basis for protein annotation. Sequence alignment tools reliably identify homologs sharing high sequence similarity. However, identification of homologs that share low sequence similarity remains a challenge. Lowering the cutoff value could enable the identification of diverged homologs, but also introduces numerous false hits. Methods are being continuously developed to minimize this problem. Estimation of the fraction of homologs in a set of protein alignments can help in the assessment and development of such methods, and provides the users with intuitive quantitative assessment of protein alignment results. Herein, we present a computational approach that estimates the amount of homologs in a set of protein pairs. The method requires a prevalent and detectable protein feature that is conserved between homologs. By analyzing the feature prevalence in a set of pairwise protein alignments, the method can estimate the number of homolog pairs in the set independently of the alignments' quality. Using the HomoloGene database as a standard of truth, we implemented this approach in a proteome-wide analysis. The results revealed that this approach, which is independent of the alignments themselves, works well for estimating the number of homologous proteins in a wide range of homology values. In summary, the presented method can accompany homology searches and method development, provides validation to search results, and allows tuning of tools and methods.

Bioinformatics, Oct 6, 2005

Motivation: Viruses and developers of anti-inflammatory therapies share a common interest in prot... more Motivation: Viruses and developers of anti-inflammatory therapies share a common interest in proteins that manipulate the immune response. Large double-stranded DNA viruses acquire host proteins to evade host defense mechanisms. Hence, viral pirated proteins may have a therapeutic potential. Although dozens of viral piracy events have already been identified, we hypothesized that sequence divergence impedes the discovery of many others. Results: We developed a method to assess the number of viral/human homologs and discovered that at least 917 highly diverged homologs are hidden in low-similarity alignment hits that are usually ignored. However, these low-similarity homologs are masked by many false alignment hits. We therefore applied a filtering method to increase the proportion of viral/human homologous proteins. The homologous proteins we found may facilitate functional annotation of viral and human proteins. Furthermore, some of these proteins play a key role in immune modulation and are therefore therapeutic protein candidates.

Journal of Chemical Information and Modeling, Mar 8, 2012

Target-oriented substructure-based virtual screening (sSBVS) of molecules is a promising approach... more Target-oriented substructure-based virtual screening (sSBVS) of molecules is a promising approach in drug discovery. Yet, there are doubts whether sSBVS is suitable also for extrapolation, that is, for detecting molecules that are very different from those used for training. Herein, we evaluate the predictive power of classic virtual screening methods, namely, similarity searching using Tanimoto coefficient (MTC) and Naive Bayes (NB). As could be expected, these classic methods perform better in interpolation than in extrapolation tasks. Consequently, to enhance the predictive ability for extrapolation tasks, we introduce the Shadow approach, in which inclusion relations between substructures are considered, as opposed to the classic sSBVS methods that assume independence between substructures. Specifically, we discard contributions from substructures included in (&amp;amp;amp;amp;amp;amp;amp;quot;shaded&amp;amp;amp;amp;amp;amp;amp;quot; by) others which are, in turn, included in the molecule of interest. Indeed, the Shadow classifier significantly outperforms both MTC (pValue = 3.1 × 10(-16)) and NB (pValue = 3.5 × 10(-9)) in detecting hits sharing low similarity with the training active molecules.

Bioinformatics, Mar 4, 2008

Motivation: Many secretory proteins are synthesized as inactive precursors that must undergo post... more Motivation: Many secretory proteins are synthesized as inactive precursors that must undergo post-translational proteolysis in order to mature and become active. In the current study, we address the challenge of sequence-based discovery of proteolytic sites in secreted proteins using machine learning. Results: The results revealed that only half of the extracellular proteolytic sites are currently annotated, leaving over 3600 unannotated ones. Furthermore, we have found that only 6% of the unannotated sites are similar to known proteolytic sites, whereas the remaining 94% do not share significant similarity with any annotated proteolytic site. The computational challenges in these two cases are very different. While the precision in detecting the former group is close to perfect, only a mere 22% of the latter group were detected with a precision of 80%. The applicability of the classifier is demonstrated through members of the FGF family, in which we verified the conservation of physiologically-relevant proteolytic sites in homologous proteins.