Protein Tyrosine Phosphatase regulation by Reactive Oxygen Species

Abstract

Protein Tyrosine Phosphatases (PTPs) help to maintain the balance of protein phosphorylation signals that drive cell division, proliferation, and differentiation. These enzymes are also well-suited to redox-dependent signaling and oxidative stress response due to their cysteine-based catalytic mechanism, which requires a deprotonated thiol group at the active site. This review focuses on PTP structural characteristics, active site chemical properties, and vulnerability to change by reactive oxygen species (ROS). PTPs can be oxidized and inactivated by H₂O₂ through three non-exclusive mechanisms. These pathways are dependent on the coordinated actions of other H₂O₂-sensitive proteins, such as peroxidases like Peroxiredoxins (Prx) and Thioredoxins (Trx). PTPs undergo reversible oxidation by converting their active site cysteine from thiol to sulfenic acid. This sulfenic acid can then react with adjacent cysteines to form disulfide bonds or with nearby amides to form sulfenyl-amide linkages. Further oxidation of the sulfenic acid form to the sulfonic or sulfinic acid forms causes irreversible deactivation. Understanding the structural changes involved in both reversible and irreversible PTP oxidation can help with their chemical manipulation for therapeutic intervention. Nonetheless, more information remains unidentified than is presently known about the precise dynamics of proteins participating in oxidation events, as well as the specific oxidation states that can be targeted for PTPs. This review summarizes current information on PTP-specific oxidation patterns and explains how ROS-mediated signal transmission interacts with phosphorylation-based signaling machinery controlled by growth factor receptors and PTPs.

Keywords: Cysteine; Oxidation; PTP; PTP1B; Phosphatase inactivation; Protein tyrosine phosphatase; ROS; Reactive oxygen species; SHP2.

Figure 1:. The PTP Superfamily of Enzymes:

A. Illustration of human PTPs with membrane-bound (receptor) and cytosolic (non-receptor) subtypes. B. (right) Domain architecture of the conserved PTP domain, indicating the positions of ten sequence motifs that are conserved across the superfamily. (left) An alternative view of the domain showing the active site’s position between numerous mobile loops. PTP1B crystal structures have been used for both the illustrations; open structure PDB ID: 5K9V, closed structure PDB ID: 1PTV.

Figure 2:. The PTP catalytic mechanism:

PTPs employ a conserved nucleophilic cysteine nested between a conserved HC-X₅-R active site motif. Stage I: The active site cysteine has a low pKa and operates in its thiolate form. Stage II: The pY-loop facilitates the entry of phosphotyrosine into the active site. The cysteine in the active site carries out a nucleophilic attack on the reactive phosphate group. This is facilitated by the aspartate residue in the WPD loop, which acts as a general acid/base. Stage III: A covalent intermediate is formed at the active site in the form of a cysteinyl phosphate group. Stage IV: glutamine from the Q loop activates water molecules to aid hydrolysis of the cysteinyl phosphate intermediate and regenerates the active site for subsequent catalytic cycles.

Figure 3:. Intracellular sources of ROS:

A. NADPH oxidase (NOX) enzymes are the primary producers of ROS. NOX enzymes are localized to several organelles and cellular membranes where they create superoxide (O₂^•−) radicals as a byproduct of NADPH oxidation. Lipoxygenases that convert arachidonic acid (2AA) to hydroperoxyeicosatetraenoic acids (HPETEs) also release superoxide (O₂^•−) into the cytosol. Subsequently, these superoxide molecules are transformed into hydrogen peroxide (H₂O₂) by the various superoxide dismutases (SOD). B. The mitochondrial respiratory chain is an incidental source of ROS. Superoxide (O₂^•−) radicals are created as a byproduct of ATP production and the movement of electrons through the electron transport chain. Included in the inner mitochondrial membrane is the respiratory chain complex comprised of NADH dehydrogenase (complex I), the succinate-coenzyme Q reductase complex (complex II), the cytochrome b-c1 complex (complex III), the cytochrome oxidase complex (complex IV) and the ATP synthase complex. Mitochondrial dismutases including MnSOD (manganese superoxide dismutase) and CuZnSOD (copper/zinc superoxide dismutase) convert superoxide (O₂^•−) radicals into the membrane permeable hydrogen peroxide (H₂O₂).

Figure 4:. PTP oxidation pathways:

In the presence of H₂O₂, the PTP catalytic cysteine’s thiolate ion is reversibly oxidized to sulfenic acid. Sulfenic acid’s instability causes it to quickly transform into a thiolate ion or reversibly oxidized intramolecular disulfide or sulfenyl amide state. Agents like glutathione and thioredoxin can reduce these to a thiol group. However, further oxidation of the sulfenic acid by H₂O₂ creates irreversibly oxidized sulfinic acid and sulfonic acid forms that permanently inactivate PTP active sites.

Figure 5:. Cellular mechanisms of PTP oxidation:

PTPs are oxidized by H₂O₂ directly, or indirectly through the oxidation of peroxiredoxin (Prx) and thioredoxin (Trx) proteins that contain reactive thiols with higher affinity for hydrogen peroxide (H₂O₂) that PTPs. In the indirect mechanism, a redox relay of thiol-disulfide exchange reactions engages several proteins that are eventually reduced by glutathione and thioredoxin.

Figure 6:. Structural features of reduced/oxidized PTP1B:

A. The reduced functional PTP1B contains a central active site Cysteine in a Thiol form flanked by various other active site loops. The WPD loop faces away from the active site cysteine in the apo (left, green) and moves towards the cysteine when the substrate binds (center, pink). The inset on the right shows the relative movements of the active site loops upon phosphotyrosine binding. B. The reversibly oxidized sulfenyl amide form of PTP1B is formed when the active site cysteine makes an “S-N” bond with the backbone nitrogen atom of the neighboring serine. This structure shows a unique and distinct conformational change in its P-loop (inset, right). C. Higher oxidized forms of PTP1B include its cysteine’s oxidation to sulfenic acid (green, left), sulfinic acid (center, blue), and sulfonic acid (right, gray). The sulfinic and sulfonic acid forms cannot be reduced by antioxidants to regenerate the active form of the enzyme. The insets under each structure show the chemical groups of the P-loop and highlight the hydrogen bonds between the active site cysteine and its neighboring residues.

Figure 7:. Reversible oxidation via disulfide binds with a backdoor cysteine:

Lyp (A, top) and SHP2 (B, bottom) use the backdoor cysteine in motif 7 (KCxxYWP) to make a reversible disulfide bond and prevent irreversible oxidization of the active site cysteine to sulfinic and sulfonic acids. The insets on the right show the active site structure and disulfide bond formation in the two PTPs.