THEMIS is a priming substrate of non-receptor tyrosine phosphatase PTPN6/SHP1 and plays dual roles during T cell development

THEMIS plays an indispensable role in T cells, but its mechanism of action is highly controversial. Using the systematic proximity labeling methodology PEPSI, we identified THEMIS as an uncharacterized substrate for the phosphatase SHP1. Saturated mutagenesis analysis revealed that THEMIS phosphorylation at the evolutionally conserved Tyr34 residue was oppositely regulated by SHP1 and the kinase LCK. Like THEMIS-/- mice, THEMIS Y34F/Y34F knock-in mice showed a significant decrease in CD4 thymocytes and mature CD4 T cells, but a normal thymic development and peripheral homeostasis of CD8 T cells. Mechanistically, phosphorylated THEMIS induced by TCR activation acts as a "priming substrate" to bind SHP1 and convert its phosphatase activity from basal level to nearly fully activated level, ensuring an appropriate negative regulation of TCR signaling. However, cytokine signaling in CD8 T cells failed to elicit THEMIS Y34 phosphorylation, revealing both phosphorylation-dependent and -independent roles of THEMIS in controlling T cell maturation and expansion.

Identification of the atypical cadherin FAT1 as a novel glypican-3 interacting protein in liver cancer cells

Glypican-3 (GPC3) is a cell surface heparan sulfate proteoglycan that is being evaluated as an emerging therapeutic target in hepatocellular carcinoma (HCC). GPC3 has been shown to interact with several extracellular signaling molecules, including Wnt, HGF, and Hedgehog. Here, we reported a cell surface transmembrane protein (FAT1) as a new GPC3 interacting protein. The GPC3 binding region on FAT1 was initially mapped to the C-terminal region (Q14517, residues 3662-4181), which covered a putative receptor tyrosine phosphatase (RTP)-like domain, a Laminin G-like domain, and five EGF-like domains. Fine mapping by ELISA and flow cytometry showed that the last four EGF-like domains (residues 4013-4181) contained a specific GPC3 binding site, whereas the RTP domain (residues 3662-3788) and the downstream Laminin G-2nd EGF-like region (residues 3829-4050) had non-specific GPC3 binding. In support of their interaction, GPC3 and FAT1 behaved concomitantly or at a similar pattern, e.g. having elevated expression in HCC cells, being up-regulated under hypoxia conditions, and being able to regulate the expression of EMT-related genes Snail, Vimentin, and E-Cadherin and promoting HCC cell migration. Taken together, our study provides the initial evidence for the novel mechanism of GPC3 and FAT1 in promoting HCC cell migration.

Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase–mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.

In Silico and in Vitro Studies of Fluorinated Chroman-2-Carboxilic Acid Derivatives as an Anti-tubercular Agent

Background: Despite the use of traditional method, Ugi reaction currently is a well-established multicomponent reaction. Chromane motif itself possesses a variety of biological functions. In order to improve its anti-tubercular activity, it is necessary to modify it accordingly. Aim: To ensure relation between in silico and in vitro study, we have carried out in vitro screening against H37Rv anti-tubercular agent. Materials and methods: Ugi four-component condensation (U-4CCRs) between 6-fluorochroman-2-carboxylic acid, various aryl aldehyde, 3,4,5-trimethoxy amine and tert-butyl isocyanide, gave N-((tert-butylcarbamoyl)(4-substitutedphenyl) methyl)-6-fluoro-N-(3,4,5-trimethoxyphenyl) chroman-2-carboxamide. The molecular level insight of all compounds was carried out by molecular docking study against the receptor tyrosine phosphatase PtpB. All these newly synthesized compounds were screened for their anti-microbial activity against Mycobacterium tuberculosis H37Rv to determine the MIC, IC50 and IC90 of the compound. Results: The compound 5d also shows large hydrophobic surface contact on the face of the α7–α8 (Ile 207, Phe 211, Met 206, Ile203, Phe161, Phe80, Met126, Tyr130, Val231 and Leu101) that lines one side of the entrance to the active site of the receptor. The compound 5d bind with tyrosine phosphatase PtpB with predicted docking geometric score of 4664, whereas a score of rifampicin was 6586 determined. Conclusion: From the docking studies, compound 5d, was considered to be the potent inhibitor, which gave strong supportive coordinate with the in vitro study. It is highly active against H37Rv, having MIC and IC50 value of was 70 μM and 53 μM respectively in in vitro study.

Loss of Ptpn11 (Shp2) drives satellite cells into quiescence

The equilibrium between proliferation and quiescence of myogenic progenitor and stem cells is tightly regulated to ensure appropriate skeletal muscle growth and repair. The non-receptor tyrosine phosphatase Ptpn11 (Shp2) is an important transducer of growth factor and cytokine signals. Here we combined complex genetic analyses, biochemical studies and pharmacological interference to demonstrate a central role of Ptpn11 in postnatal myogenesis of mice. Loss of Ptpn11 drove muscle stem cells out of the proliferative and into a resting state during muscle growth. This Ptpn11 function was observed in postnatal but not fetal myogenic stem cells. Furthermore, muscle repair was severely perturbed when Ptpn11 was ablated in stem cells due to a deficit in stem cell proliferation and survival. Our data demonstrate a molecular difference in the control of cell cycle withdrawal in fetal and postnatal myogenic stem cells, and assign to Ptpn11 signaling a key function in satellite cell activity.

Fat2 acts through the WAVE regulatory complex to drive collective cell migration during tissue rotation

Directional cell movements during morphogenesis require the coordinated interplay between membrane receptors and the actin cytoskeleton. The WAVE regulatory complex (WRC) is a conserved actin regulator. Here, we found that the atypical cadherin Fat2 recruits the WRC to basal membranes of tricellular contacts where a new type of planar-polarized whip-like actin protrusion is formed. Loss of either Fat2 function or its interaction with the WRC disrupts tricellular protrusions and results in the formation of nonpolarized filopodia. We provide further evidence for a molecular network in which the receptor tyrosine phosphatase Dlar interacts with the WRC to couple the extracellular matrix, the membrane, and the actin cytoskeleton during egg elongation. Our data uncover a mechanism by which polarity information can be transduced from a membrane receptor to a key actin regulator to control collective follicle cell migration during egg elongation. 4D-live imaging of rotating MCF10A mammary acini further suggests an evolutionary conserved mechanism driving rotational motions in epithelial morphogenesis.