Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B

Abstract

Protein tyrosine phosphatases regulate signal transduction pathways involving tyrosine phosphorylation and have been implicated in the development of cancer, diabetes, rheumatoid arthritis and hypertension. Increasing evidence suggests that the cellular redox state is involved in regulating tyrosine phosphatase activity through the reversible oxidization of the catalytic cysteine to sulphenic acid (Cys-SOH). But how further oxidation to the irreversible sulphinic (Cys-SO2H) and sulphonic (Cys-SO3H) forms is prevented remains unclear. Here we report the crystal structures of the regulatory sulphenic and irreversible sulphinic and sulphonic acids of protein tyrosine phosphatase 1B (PTP1B), an important enzyme in the negative regulation of the insulin receptor and a therapeutic target in type II diabetes and obesity. We also identify a sulphenyl-amide species that is formed through oxidation of its catalytic cysteine. Formation of the sulphenyl-amide causes large changes in the PTP1B active site, which are reversible by reduction with the cellular reducing agent glutathione. The sulphenyl-amide is a protective intermediate in the oxidative inhibition of PTP1B. In addition, it may facilitate reactivation of PTP1B by biological thiols and signal a unique state of the protein.

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…One of the mechanisms involves the reaction of cysteine residue oxidized to sulfenic acid with an amide from the protein backbone. This reaction has been observed in purified proteins and in absence of glutathione in several PTPs (27,75). Thus, while feasible, this reaction has not yet been demonstrated to occur in vivo.…”

“…Apart from glutathionylation, another chemical modification to prevent cysteine overoxidation has recently been discovered in the form of sulfenyl-amide formation. It was shown that, the cysteine residue in protein tyrosine phosphatase 1B (PTP1B) can react after oxidation to a sulfenic acid by H 2 O 2 in the absence of GSH (26,27). The sulfenic acid reacts further with an amide nitrogen of the backbone to form a cyclic sulfenyl-amide.…”

“…Those proteins are included here as the same critical cysteine(s) can be modified by either ROS or RNS and very likely by HNE as well when the cysteine is in the thiolate form. Among the signaling proteins (and their critical cysteines) that have been well characterized are the PTPs, PTP1B (cys 215 ) (26,27,(34)(35)(36), low molecular weight PTP (cys12,17) (37-39) and Srchomology 2 domain-containing PTP (SHP-2) (40), the small G protein, Ras (cys 118 ) (41)(42)(43)(44), the large G proteins, G i (cys 267 ) and G o (cys 326 ) (45) and the lipid phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In addition, as mentioned above, in its reduced form Trx binds and inhibits ASK1 and possibly other signaling proteins as well (46)(47)(48)(49)(50)(51)(52)(53).…”

“…The cysteine thiolate of PTP1B can be readily oxidized to sulfenic acid, a reversible form of oxidation, but can be oxidized further to a sulfinic or sulfonic acid (27). As described above, Srx can reduce the sulfinic acid form of Prdx 1 (71) but thus far no evidence exists for the formation or reduction of the sulfinic acid form of PTP1B in vivo.…”

“…Although the mechanism by which glutathionylation occurs is not agreed upon, this process has been shown occur in vivo in relevant cell types (81)(82)(83)(84)(85)(86). Although glutathionylation of PTP1B can be achieved in vitro with the purified enzyme through oxidation by H 2 O 2 and addition of GSH (through the sulfenic acid and sulfenyl-amide as described above) (26) (27) or by addition of a very high concentration of GSSG (63), it is not known if glutathionylation occurs enzymatically or non-enzymatically in vivo and kinetic considerations clearly argue against the latter (87). While formation of glutathionylated PTP1B through disulfide exchange in the cytosol by reaction with GSSG enzymatically or non-enzymatically is unlikely due to the requirement for markedly increasing GSSG, a recent paper by Beer et al demonstrated glutaredoxin 2 dependent catalysis of glutathionylation/deglutationylation of mitochondrial proteins (88).…”