10 results on '"Sachdev S Sidhu"'
Search Results
2. Peptide binding properties of the three PDZ domains of Bazooka (Drosophila Par-3).
- Author
-
Cao Guo Yu, Raffi Tonikian, Corinna Felsensteiner, Jacquelyn R Jhingree, Darrell Desveaux, Sachdev S Sidhu, and Tony J C Harris
- Subjects
Medicine ,Science - Abstract
The Par complex is a conserved cell polarity regulator. Bazooka/Par-3 is scaffold for the complex and contains three PDZ domains in tandem. PDZ domains can act singly or synergistically to bind the C-termini of interacting proteins. Sequence comparisons among Drosophila Baz and its human and C. elegans Par-3 counterparts indicate a divergence of the peptide binding pocket of PDZ1 and greater conservation for the pockets of PDZ2 and PDZ3. However, it is unclear whether the domains from different species share peptide binding preferences, or if their tandem organization affects their peptide binding properties. To investigate these questions, we first used phage display screens to identify unique peptide binding profiles for each single PDZ domain of Baz. Comparisons with published phage display screens indicate that Baz and C. elegans PDZ2 bind to similar peptides, and that the peptide binding preferences of Baz PDZ3 are more similar to C. elegans versus human PDZ3. Next we quantified the peptide binding preferences of each Baz PDZ domain using single identified peptides in surface plasmon resonance assays. In these direct binding studies, each peptide had a binding preference for a single PDZ domain (although the peptide binding of PDZ2 was weakest and the least specific). PDZ1 and PDZ3 bound their peptides with dissociation constants in the nM range, whereas PDZ2-peptide binding was in the µM range. To test whether tandem PDZ domain organization affects peptide binding, we examined a fusion protein containing all three PDZ domains and their normal linker regions. The binding strengths of the PDZ-specific peptides to single PDZ domains and to the PDZ domain tandem were indistinguishable. Thus, the peptide binding pockets of each PDZ domain in Baz are not obviously affected by the presence of neighbouring PDZ domains, but act as isolated modules with specific in vitro peptide binding preferences.
- Published
- 2014
- Full Text
- View/download PDF
3. Prediction and experimental characterization of nsSNPs altering human PDZ-binding motifs.
- Author
-
David Gfeller, Andreas Ernst, Nick Jarvik, Sachdev S Sidhu, and Gary D Bader
- Subjects
Medicine ,Science - Abstract
Single nucleotide polymorphisms (SNPs) are a major contributor to genetic and phenotypic variation within populations. Non-synonymous SNPs (nsSNPs) modify the sequence of proteins and can affect their folding or binding properties. Experimental analysis of all nsSNPs is currently unfeasible and therefore computational predictions of the molecular effect of nsSNPs are helpful to guide experimental investigations. While some nsSNPs can be accurately characterized, for instance if they fall into strongly conserved or well annotated regions, the molecular consequences of many others are more challenging to predict. In particular, nsSNPs affecting less structured, and often less conserved regions, are difficult to characterize. Binding sites that mediate protein-protein or other protein interactions are an important class of functional sites on proteins and can be used to help interpret nsSNPs. Binding sites targeted by the PDZ modular peptide recognition domain have recently been characterized. Here we use this data to show that it is possible to computationally identify nsSNPs in PDZ binding motifs that modify or prevent binding to the proteins containing the motifs. We confirm these predictions by experimentally validating a selected subset with ELISA. Our work also highlights the importance of better characterizing linear motifs in proteins as many of these can be affected by genetic variations.
- Published
- 2014
- Full Text
- View/download PDF
4. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.
- Author
-
Keith R Miller, Akiko Koide, Brenda Leung, Jonathan Fitzsimmons, Bryan Yoder, Hong Yuan, Michael Jay, Sachdev S Sidhu, Shohei Koide, and Edward J Collins
- Subjects
Medicine ,Science - Abstract
A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64)Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.
- Published
- 2012
- Full Text
- View/download PDF
5. Charged and hydrophobic surfaces on the a chain of shiga-like toxin 1 recognize the C-terminal domain of ribosomal stalk proteins.
- Author
-
Andrew J McCluskey, Eleonora Bolewska-Pedyczak, Nick Jarvik, Gang Chen, Sachdev S Sidhu, and Jean Gariépy
- Subjects
Medicine ,Science - Abstract
Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A(1) chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A(1) chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A(1) chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A(1) variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A(1) chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A(1) chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A(1) chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs.
- Published
- 2012
- Full Text
- View/download PDF
6. Prediction and experimental characterization of nsSNPs altering human PDZ-binding motifs
- Author
-
Sachdev S. Sidhu, Nick Jarvik, David Gfeller, Gary D. Bader, and Andreas Ernst
- Subjects
Proteomics ,Protein Structure ,Molecular Sequence Data ,PDZ domain ,PDZ Domains ,lcsh:Medicine ,Single-nucleotide polymorphism ,Plasma protein binding ,Biology ,Polymorphism, Single Nucleotide ,Biochemistry ,Protein–protein interaction ,Databases, Genetic ,Genetics ,Macromolecular Structure Analysis ,Humans ,Short linear motif ,Amino Acid Sequence ,Binding site ,Protein Interactions ,Molecular Biology Techniques ,Sequencing Techniques ,lcsh:Science ,Molecular Biology ,Peptide sequence ,Macromolecular Complex Analysis ,Binding Sites ,Models, Statistical ,Multidisciplinary ,Genome, Human ,Systems Biology ,lcsh:R ,Proteins ,Biology and Life Sciences ,Computational Biology ,Genomics ,Functional Genomics ,Mutation ,Human genome ,lcsh:Q ,Sequence Analysis ,Protein Binding ,Research Article - Abstract
Single nucleotide polymorphisms (SNPs) are a major contributor to genetic and phenotypic variation within populations. Non-synonymous SNPs (nsSNPs) modify the sequence of proteins and can affect their folding or binding properties. Experimental analysis of all nsSNPs is currently unfeasible and therefore computational predictions of the molecular effect of nsSNPs are helpful to guide experimental investigations. While some nsSNPs can be accurately characterized, for instance if they fall into strongly conserved or well annotated regions, the molecular consequences of many others are more challenging to predict. In particular, nsSNPs affecting less structured, and often less conserved regions, are difficult to characterize. Binding sites that mediate protein-protein or other protein interactions are an important class of functional sites on proteins and can be used to help interpret nsSNPs. Binding sites targeted by the PDZ modular peptide recognition domain have recently been characterized. Here we use this data to show that it is possible to computationally identify nsSNPs in PDZ binding motifs that modify or prevent binding to the proteins containing the motifs. We confirm these predictions by experimentally validating a selected subset with ELISA. Our work also highlights the importance of better characterizing linear motifs in proteins as many of these can be affected by genetic variations.
- Published
- 2014
7. Peptide Binding Properties of the Three PDZ Domains of Bazooka (Drosophila Par-3)
- Author
-
Darrell Desveaux, Tony J. C. Harris, Jacquelyn R. Jhingree, Corinna Felsensteiner, Cao Guo Yu, Sachdev S. Sidhu, and Raffi Tonikian
- Subjects
Protein Structure ,Phage display ,Amino Acid Motifs ,Molecular Sequence Data ,Protein domain ,PDZ domain ,lcsh:Medicine ,PDZ Domains ,Peptide binding ,Peptide ,Plasma protein binding ,Biology ,Biochemistry ,Model Organisms ,Animals ,Drosophila Proteins ,Humans ,Position-Specific Scoring Matrices ,Amino Acid Sequence ,Biomacromolecule-Ligand Interactions ,lcsh:Science ,Protein Interactions ,Peptide sequence ,Conserved Sequence ,chemistry.chemical_classification ,Multidisciplinary ,Drosophila Melanogaster ,lcsh:R ,Intracellular Signaling Peptides and Proteins ,Proteins ,Animal Models ,Molecular biology ,Fusion protein ,Recombinant Proteins ,Regulatory Proteins ,chemistry ,Biophysics ,lcsh:Q ,Drosophila ,Structural Proteins ,Peptides ,Sequence Alignment ,Research Article ,Protein Binding - Abstract
The Par complex is a conserved cell polarity regulator. Bazooka/Par-3 is scaffold for the complex and contains three PDZ domains in tandem. PDZ domains can act singly or synergistically to bind the C-termini of interacting proteins. Sequence comparisons among Drosophila Baz and its human and C. elegans Par-3 counterparts indicate a divergence of the peptide binding pocket of PDZ1 and greater conservation for the pockets of PDZ2 and PDZ3. However, it is unclear whether the domains from different species share peptide binding preferences, or if their tandem organization affects their peptide binding properties. To investigate these questions, we first used phage display screens to identify unique peptide binding profiles for each single PDZ domain of Baz. Comparisons with published phage display screens indicate that Baz and C. elegans PDZ2 bind to similar peptides, and that the peptide binding preferences of Baz PDZ3 are more similar to C. elegans versus human PDZ3. Next we quantified the peptide binding preferences of each Baz PDZ domain using single identified peptides in surface plasmon resonance assays. In these direct binding studies, each peptide had a binding preference for a single PDZ domain (although the peptide binding of PDZ2 was weakest and the least specific). PDZ1 and PDZ3 bound their peptides with dissociation constants in the nM range, whereas PDZ2-peptide binding was in the µM range. To test whether tandem PDZ domain organization affects peptide binding, we examined a fusion protein containing all three PDZ domains and their normal linker regions. The binding strengths of the PDZ-specific peptides to single PDZ domains and to the PDZ domain tandem were indistinguishable. Thus, the peptide binding pockets of each PDZ domain in Baz are not obviously affected by the presence of neighbouring PDZ domains, but act as isolated modules with specific in vitro peptide binding preferences.
- Published
- 2014
8. T Cell Receptor-Like Recognition of Tumor In Vivo by Synthetic Antibody Fragment
- Author
-
Edward J. Collins, Hong Yuan, Bryan C. Yoder, Sachdev S. Sidhu, Akiko Koide, Michael Jay, Brenda Leung, Keith R. Miller, Jonathan Fitzsimmons, and Shohei Koide
- Subjects
Radionuclide imaging ,Receptor, ErbB-2 ,Cancer Treatment ,PET imaging ,Antigen Processing and Recognition ,Plasma protein binding ,Major Histocompatibility Complex ,Mice ,0302 clinical medicine ,Cricetinae ,Receptor ,Immunoglobulin Fragments ,0303 health sciences ,Multidisciplinary ,3. Good health ,Synthetic antibody ,Oncology ,Medicine ,Antibody ,Radiology ,Research Article ,Protein Binding ,Tumor Immunology ,Science ,Immunology ,Receptors, Antigen, T-Cell ,CHO Cells ,Biology ,Major histocompatibility complex ,03 medical and health sciences ,Immune system ,Antibody Therapy ,Antigen ,Cell Line, Tumor ,HLA-A2 Antigen ,Cancer Detection and Diagnosis ,Animals ,Humans ,030304 developmental biology ,T-cell receptor ,Immunologic Subspecialties ,Molecular biology ,Nuclear medicine ,biology.protein ,Clinical Immunology ,Peptides ,030215 immunology - Abstract
A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64)Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.
- Published
- 2012
9. Charged and Hydrophobic Surfaces on the A Chain of Shiga-Like Toxin 1 Recognize the C-Terminal Domain of Ribosomal Stalk Proteins
- Author
-
Sachdev S. Sidhu, Jean Gariépy, Eleonora Bolewska-Pedyczak, Andrew J. McCluskey, Gang Chen, and Nick Jarvik
- Subjects
Ribosomal Proteins ,Molecular Sequence Data ,Protein domain ,lcsh:Medicine ,Tripeptide ,Shiga Toxin 1 ,Toxicology ,Biochemistry ,Microbiology ,Ribosome ,03 medical and health sciences ,Ribosomal protein ,Catalytic Domain ,Two-Hybrid System Techniques ,Amino Acid Sequence ,lcsh:Science ,Biology ,Peptide sequence ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Sequence Homology, Amino Acid ,Tetrapeptide ,030306 microbiology ,Chemistry ,C-terminus ,lcsh:R ,Surface Plasmon Resonance ,Ribosomal RNA ,Recombinant Proteins ,Protein Structure, Tertiary ,Bacterial Pathogens ,RNA, Ribosomal ,Protein Biosynthesis ,Medicine ,lcsh:Q ,Hydrophobic and Hydrophilic Interactions ,Oligopeptides ,Ribosomes ,Research Article - Abstract
Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A(1) chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A(1) chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A(1) chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A(1) variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A(1) chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A(1) chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A(1) chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs.
- Published
- 2012
10. Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli.
- Author
-
Luz D, Chen G, Maranhão AQ, Rocha LB, Sidhu S, and Piazza RM
- Subjects
- Animals, Cell Line, Humans, Hybridomas immunology, Mice, Peptide Library, Sensitivity and Specificity, Shiga-Toxigenic Escherichia coli immunology, Single-Chain Antibodies genetics, Antibodies, Neutralizing metabolism, Immunoglobulin Fab Fragments metabolism, Shiga Toxin 2 antagonists & inhibitors, Shiga-Toxigenic Escherichia coli metabolism, Single-Chain Antibodies metabolism
- Abstract
Background: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes., Methods and Findings: In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA., Conclusion: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.
- Published
- 2015
- Full Text
- View/download PDF
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