Characterization of sabatolimab, a novel immunotherapy with immuno-myeloid activity directed against TIM-3 receptor.

Objectives: Sabatolimab is a humanized monoclonal antibody (hIgG4, S228P) directed against human T-cell immunoglobulin domain and mucin domain-3 (TIM-3). Herein, we describe the development and characterization of sabatolimab.

Methods: Sabatolimab was tested for binding to its target TIM-3 and blocking properties.

Whole grain in manufactured foods: Current use, challenges and the way forward.

Some countries now incorporate recommendations for increased consumption of whole grain (WG) into local dietary guidelines. Cereal and pseudo-cereal grains are good sources of complex carbohydrates, dietary fiber, proteins, phytochemicals, vitamins and minerals. However, research shows that the large majority of consumers are still falling short of WG consumption goals.

LyGDI, a novel SHIP-interacting protein, is a negative regulator of FcγR-mediated phagocytosis.

SHIP and SHIP-2 are inositol phosphatases that regulate FcγR-mediated phagocytosis through catalytic as well as non-catalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE) analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon FcγR clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex.

Substrate specificity of protein tyrosine phosphatases 1B, RPTPα, SHP-1, and SHP-2.

We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately 2 orders of magnitude less active than the other three PTPs.

The SH2 domains of inositol polyphosphate 5-phosphatases SHIP1 and SHIP2 have similar ligand specificity but different binding kinetics.

SH2 domain-containing inositol 5-phosphatases 1 (SHIP1) and 2 (SHIP2) are structurally similar proteins that catalyze the degradation of lipid secondary messenger phosphatidylinositol 3,4,5-triphosphate to produce phosphatidylinositol 3,4-diphosphate. Despite their high sequence identity (51%), SHIP1 and SHIP2 share little overlap in their in vivo functions. In this work, the sequence specificity of the SHIP2 SH2 domain was systematically defined through the screening of a combinatorial pY peptide library.

Synthesis of 3,5-difluorotyrosine-containing peptides: application in substrate profiling of protein tyrosine phosphatases.

Fully protected 3,5-difluorotyrosine (F2Y), Fmoc-F2Y(tBu)-OH, is efficiently prepared by a chemoenzymatic process and incorporated into individual peptides and combinatorial peptide libraries. The F2Y-containing peptides display kinetic properties toward protein tyrosine phosphatases (PTPs) similar to their corresponding tyrosine-containing counterparts but are resistant to tyrosinase action. These properties make F2Y a useful tyrosine surrogate during peptide library screening for optimal PTP substrates.

Cyclic peptidyl inhibitors of Grb2 and tensin SH2 domains identified from combinatorial libraries.

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. In this work, a novel method has been developed for the high-throughput synthesis, screening, and identification of cyclic peptidyl ligands against macromolecular targets. Support-bound cyclic phosphotyrosyl peptide libraries containing randomized amino acid sequences and different ring sizes (theoretical diversity of 3.

Reverse interactomics: decoding protein-protein interactions with combinatorial peptide libraries.

Identification of binding partners is the crucial first step towards understanding the biological function of a protein. Many protein-protein interactions occur via modular domains that recognize short peptide motifs in their target proteins. Here we describe a chemical/bioinformatics approach for predicting the binding partners of modular domains.

Defining SH2 domain and PTP specificity by screening combinatorial peptide libraries.

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing short phosphotyrosyl (pY) peptide motifs in their partner proteins. Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of pY proteins, counteracting the protein tyrosine kinases. Both types of proteins exhibit primary sequence specificity, which plays at least a partial role in dictating their physiological interacting partners or substrates.

A chemical approach to the identification of tensin-binding proteins.

Many protein-protein interactions are mediated by small modular domains, which recognize short peptide motifs in their partner proteins. However, for the great majority of these domains, the identity of their partner proteins remains unknown. In this work, a chemical/bioinformatics approach has been developed to identify phosphotyrosyl (pY) proteins that bind to tensin, a protein involved in the formation of actin cytoskeleton and signal transduction.

Traceless capping agent for peptide sequencing by partial edman degradation and mass spectrometry.

An improved method for the rapid sequence determination of biologically active peptides selected from one-bead-one-peptide combinatorial libraries has been developed. In this method, beads carrying unique peptide sequences were subjected to multiple cycles of partial Edman degradation (PED) by the treatment with a 15-30:1 mixture of phenyl isothiocyanate and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSU), to generate a series of sequence-specific truncation products (a peptide ladder) for each resin-bound peptide. Following PED, the Fmoc group was removed from the N-terminus and any reacted side chains by piperidine treatment.

Sequence specificity of SHP-1 and SHP-2 Src homology 2 domains. Critical roles of residues beyond the pY+3 position.

A combinatorial phosphotyrosyl (pY) peptide library was screened to determine the amino acid preferences at the pY+4 to pY+6 positions for the four SH2 domains of protein-tyrosine phosphatases SHP-1 and SHP-2. Individual binding sequences selected from the library were resynthesized and their binding affinities and specificities to various SH2 domains were further evaluated by SPR studies, stimulation of SHP-1 and SHP-2 phosphatase activity, and in vitro pulldown assays. These studies reveal that binding of a pY peptide to the N-SH2 domain of SHP-2 is greatly enhanced by a large hydrophobic residue (Trp, Tyr, Met, or Phe) at the pY+4 and/or pY+5 positions, whereas binding to SHP-1 N-SH2 domain is enhanced by either hydrophobic or positively charged residues (Arg, Lys, or His) at these positions.

Decoding protein-protein interactions through combinatorial chemistry: sequence specificity of SHP-1, SHP-2, and SHIP SH2 domains.

A general, combinatorial library method for the rapid identification of high-affinity peptide ligands of protein modular domains is reported. The validity of this method has been demonstrated by determining the sequence specificity of four Src homology 2 (SH2) domains derived from protein tyrosine phosphatase SHP-1 and SHP-2 and inositol phosphatase SHIP. A phosphotyrosyl (pY) peptide library was screened against the SH2 domains, and the beads that carry high-affinity ligands of the SH2 domains were identified and peptides were sequenced by partial Edman degradation and mass spectrometry.

Alternative mode of binding to phosphotyrosyl peptides by Src homology-2 domains.

Src homology-2 (SH2) domains recognize specific phosphotyrosyl (pY) proteins and promote protein-protein interactions. In their classical binding mode, the SH2 domain makes specific contacts with the pY residue and the three residues immediately C-terminal to the pY, although for a few SH2 domains, residues N-terminal to pY have recently been shown to also contribute to the overall binding affinity and specificity. In this work, the ability of an SH2 domain to bind to the N-terminal side of pY has been systematically examined.

S-Ribosylhomocysteinase (LuxS) is a mononuclear iron protein.

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce L-homocysteine and 4,5-dihydroxy-2,3-pentanedione (DHPD). This is a key step in the biosynthetic pathway of the type II autoinducer (AI-2) in both Gram-positive and Gram-negative bacteria. Previous studies demonstrated that LuxS contains a divalent metal cofactor, which has been proposed to be a Zn(2+) ion.