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Development of a Peptide Inhibitor Targeting the C-SH2 Domain of the SHP2 Phosphatase

ABSTRACT: Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) has important cellular functions in mediating signal transduction downstream of receptor tyrosine kinases and immune cell receptors. In T cells, SHP2 is a key regulator of signalling pathways downstream of the checkpoint receptor programmed cell death protein 1 (PD-1) that suppresses T cell activation, and of the T cell receptor (TCR) that promotes T cell activation. SHP2 contains two SH2 domains: the N-SH2 and C-SH2 domains. Here, we describe a new approach for the chemical targeting of SHP2 by developing a peptide inhibitor, containing a non-hydrolysable phosphotyrosine (pTyr) mimetic, which selectively binds to the C-SH2 domain of SHP2. We found that incorporation of the pTyr mimetic L-O-malonyltyrosine (L-OMT) displayed robust binding affinity to the C-SH2 domain, while the widely used pTyr mimetic phosphonodifluoromethyl phenylalanine (F2Pmp) abolished binding, showing that the use of pTyr mimetics for SH2 domains is case dependent. Our C-SH2 inhibitor peptide (CSIP) impedes SHP2 activation and inhibits SHP2 phosphatase activity in vitro. CSIP is stable, cell permeable and non-cytotoxic in a model T cell line (Jurkat), in which it displays cellular activity in modulating T cell activation. As such, CSIP is a powerful chemical tool to study SHP2, in particular the role of its C-SH2 domain, in cellular contexts. CSIP enriches the toolbox of inhibitors with different modes of action to study SHP2.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Cell Culture

SUBMITTER: Julian Bender

LAB HEAD: Bettina Warscheid

PROVIDER: PXD054302 | Pride | 2025-05-09

REPOSITORIES: Pride

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Project description:Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered a hallmark in cellular signal transduction. However, its analysis remains a challenge. The conventional immunopurification (IP) approach using antibodies pan-specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we aim to optimize the approach using SH2 superbinder for tyrosine phosphoproteome analysis. The present work covalently immobilized SH2 superbinders to agrose beads, designed and evaluated four different SH2 superbinder based experimental workflows for phosphotyrosine analysis. After made a head to head comparison of them, we observed that the combined strategy employing Ti4+-IMAC and covalently immobilized SH2 superbinder enrichment in sequence generated the largest pTyr peptide dataset (3519) with the best selectivity (90%). Next the optimal method was applied for pTyr profiling from normal mouse tissues, and resulted in the identification of 197 pTyr sites (of which 73 were novel) from 5 mg protein digests, which validated its high sensitivity. An comparison with antibody 4G10 by isolating pTyr peptides from 5 mg unstimulated Jurkat cell digests were made, our optimal strategy identified 343 while the antibody 4G10 based method identified 242 pTyr sites, respectively, further confirmed the robustness of the method. Finally, the optimal strategy was applied for quantitative pTyr phosphorylation analysis of EGF stimulated/unstimulated HeLa cells, 261 pTyr sites were successfully quantified from 5 mg stable-isotope dimethyl labling protein digests. In general, the combination of Ti4+-IMAC and SH2 superbinder enrichment in sequence generated a facial and robust strategy for qualitative and quantitative analysis of pTyr proteome.

Project description:The protein modules known as SH2 (Src-homology-2) domains are key players in the signal transduction of animals. Two questions arise: Do such modules exist in plants, and when did SH2 domains evolve? Here I show that the Arabidopsis genome contains three strong candidates for plant SH2 proteins (referred to as PASTA1, 2 and 3 : GI:25513455, At1g78540, At1g17040 respectively) with homology to the SH2 domains and the adjacent linker region of STAT proteins (Signal Transducer and Activator of Transcription). The three characteristics features of a STAT protein sequence1, namely, (i) the SH2 domain with a conserved arginine residue crucial for binding to a phospho-tyrosine residue (ii) a tyrosine residue outside the C-terminus of the SH2-domain for phosphorylation during signalling and (iii) a DNA-binding domain, are conserved in the PASTA3 protein. However, PASTA 1 and 2 proteins lack a tyrosine in a similar position. PASTA proteins are not homologous to STAT proteins outside the SH2 and linker regions. The three PASTA proteins are 70 to 80 % identical to one another. Gene expression studies with PASTA2 reveal that it is expressed in roots, stem, leaves, flowers and green siliques. Preliminary indications are that plants homozygous for PASTA2 do not have any obvious phenotype, most likely due to redundancies. This microarray experiment is an attempt to compare the gene expression of a mutant plant homozygous for PASTA2 with that of the wild type plant. This might give clues about the possible function of PASTA2 in Arabidopsis. Experimenter name = Latha Kadalayil; Experimenter phone = 023-8059 5512; Experimenter department = University of Southampton; Experimenter address = School of Biological Sciences; Experimenter address = Univ. Southampton; Experimenter address = Bassett Crescent East; Experimenter address = Southampton; Experimenter zip/postal_code = SO16 7PX; Experimenter country = UK Experiment Overall Design: 2 samples were used in this experiment

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Development of a Peptide Inhibitor Targeting the C-SH2 Domain of the SHP2 Phosphatase

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