Target-specific control of lymphoid-specific protein tyrosine phosphatase (Lyp) activity

Abstract

Lymphoid-specific protein tyrosine phosphatase (Lyp), a member of the protein tyrosine phosphatase (PTP) superfamily of enzymes, is an important mediator of human-leukocyte signaling. Lyp has also emerged as a potential anti-autoimmune therapeutic target, owing to the association of a Lyp-activating mutation with an array of autoimmune disorders. Toward the goal of generating a selective inhibitor of Lyp activity that could be used for investigating Lyp's roles in cell signaling and autoimmune-disease progression, here we report that Lyp's PTP domain can be readily sensitized to target-specific inhibition by a cell-permeable small molecule. Insertion of a tetracysteine-motif-containing peptide at a conserved position in Lyp's catalytic domain generated a mutant enzyme (Lyp-CCPGCC) that retains activity comparable to that of wild-type Lyp in the absence of added ligand. Upon addition of a tetracysteine-targeting biarsenical compound (FlAsH), however, the activity of the Lyp-CCPGCC drops dramatically, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. We show that FlAsH-induced Lyp-CCPGCC inhibition is potent, specific, rapid, and independent of the nature of the PTP substrate used in the inhibition assay. Moreover, we show that FlAsH can be used to specifically target overexpressed Lyp-CCPGCC in a complex proteomic mixture. Since the mammalian-cell permeability of FlAsH is well established, it is likely that FlAsH-mediated inhibition of Lyp-CCPGCC will be useful for specifically targeting Lyp activity in engineered leukocytes and autoimmune-disease models.

Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

“…To more closely investigate potential differences in the strength between the biarsenical/dicysteine (P87C) and biarsenical/tricysteine (P87C/A122C) interactions, we subjected the PTP1B variants to further experimental challenges, analogous to those presented earlier for V368C Shp2 (Figure 7). 22 In an SDS-PAGE/fluorescence experiment, we found that only P87C/A122C PTP1B produces a strongly fluorescent band at 45 kD (the molecular weight of our PTP1B construct), whereas wild-type PTP1B and P87C PTP1B both fail to produce strongly fluorescent bands (Figure 7A). Similarly, a β-ME-challenge experiment indicated that the additional A122C mutation permits the target site in PTP1B to compete more effectively with free thiols in solution for binding with FlAsH (Figure 7B).…”

“…19–20 Targeting engineered cysteine-enriched WPD loops with biarsenicals has proven to be a general approach for PTP inhibition. 16, 21–22 However, the WPD loop constitutes part of the PTP active site (in its closed state) and plays a key role in the catalytic mechanism of PTPs, presenting the possibility that mutations on the loop itself could alter the inherent kinetic activity or substrate specificity of the enzyme. Moreover, the presence of catalytically essential residues on the WPD loop significantly constrains the options for its engineering in a manner that is functionally silent ( e.g.…”

Arsenic Binding to Proteins

“…Arsenic may inactivate up to 200 enzymes. , Most of the enzyme inhibition studies used iAs III and some used organic arsenicals, particularly phenylarsine oxide (PAO). Enzymes that were shown to be inhibited by arsenic include glutathione reductase, ,− glutathione S -transferase, glutathione peroxidase, thioredoxin reductase, − thioredoxin peroxidase, DNA ligases, Arg-tRNA protein transferase, , trypanothione reductase, IκB kinase β (IKKβ), pyruvate kinase galectin 1, protein tyrosine phosphatase, − JNK phosphatase, Wip1 phosphatase, and E3 ligases c-CBL and SIAH1 …”

Exploration of Biarsenical Chemistry—Challenges in Protein Research

“…Recently a FlAsH‐EDT 2 sensitive mutant of lymphoid‐specific PTP was developed. A nonlinear relationship between k obs and inhibitor concentration suggested a two‐step model of inhibition 57. FlAsH‐EDT 2 was also used to mimic the inhibitory effect of cadmium (Cd II ) on engineered mutants of a renal Na + /glucose co‐transporter; this provided evidence that endogenous CXXC is responsible for transporter sensitivity toward Cd II 138…”