Navasona Krishnan

H2S-Induced Sulfhydration of the Phosphatase PTP1B and Its Role in the Endoplasmic Reticulum Stress Response

Reversible inactivation by sulfhydration of the protein tyrosine phosphatase PTP1B activates the kinase PERK to promote the ER stress response. Inactivating a Phosphatase with H2S Protein tyrosine phosphatases (PTPs) have a cysteine in their active sites that is susceptible to oxidation. Krishnan et al. show that the reactive gas hydrogen sulfide (H2S) is produced by cystathionine γ-lyase during the unfolded protein response (UPR) that occurs when cells experience endoplasmic reticulum (ER) stress. In vitro experiments and experiments with cultured cells showed that H2S reacted with the active-site cysteine of PTP1B and inhibited the activity of this ER-associated enzyme. Inhibition of PTP1B by H2S during the ER stress response promoted the activity of the kinase PERK, a mediator of the UPR that inhibits protein translation. Thus, reversible posttranslational sulfhydration of PTP1B contributes to the ER stress response to promote a return to cellular homeostasis. Although originally considered toxic, hydrogen sulfide (H2S) has been implicated in mediating various biological processes. Nevertheless, its cellular targets and mode of action are not well understood. Protein tyrosine phosphatases (PTPs), which regulate numerous signal transduction pathways, use an essential cysteine residue at the active site, which is characterized by a low pKa and is susceptible to reversible oxidation. Here, we report that PTP1B was reversibly inactivated by H2S, in vitro and in cells, through sulfhydration of the active-site cysteine residue. Unlike oxidized PTP1B, the sulfhydrated enzyme was preferentially reduced in vitro by thioredoxin, compared to glutathione or dithiothreitol. Sulfhydration of PTP1B in cells required the presence of cystathionine γ-lyase (CSE), a critical enzyme in H2S production, and resulted in inhibition of phosphatase activity. Suppression of CSE decreased H2S production and decreased the phosphorylation of tyrosine-619 in PERK [protein kinase–like endoplasmic reticulum (ER) kinase], thus reducing its activation in response to ER stress. PERK, which phosphorylates the eukaryotic translational initiation factor 2, leading to attenuation of protein translation, was a direct substrate of PTP1B. In addition, CSE knockdown led to activation of the nonreceptor tyrosine kinase SRC, previously shown to be mediated by PTP1B. These effects of suppressing H2S production on the response to ER stress were abrogated by a small-molecule inhibitor of PTP1B. Together, these data define a signaling function for H2S in inhibiting PTP1B activity and thereby promoting PERK activity during the response to ER stress.

Targeting the disordered C terminus of PTP1B with an allosteric inhibitor.

PTP1B, a validated therapeutic target for diabetes and obesity, plays a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We defined a novel mechanism of allosteric inhibition that targets the C-terminal, non-catalytic segment of PTP1B. We present the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small molecule inhibitor, MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.

Dephosphorylation of the C-terminal tyrosyl residue of the DNA damage-related histone H2A.X is mediated by the protein phosphatase eyes absent.

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.

Reactivation of oxidized PTP1B and PTEN by thioredoxin 1

The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase‐dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of colocalized NADPH oxidases and is mediated by locally produced reactive oxygen species, in particular H2O2. How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual‐specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of thioredoxin 1(TRX1) slows PTEN reactivation in intact living cells. Finally, using a mechanism‐based trapping approach, we demonstrate direct thiol disulphide exchange between the active sites of thioredoxin and either phosphatase. The application of thioredoxin trapping mutants represents a complementary approach to direct assays of PTP oxidation in elucidating the significance of redox regulation of PTP function in the control of cell signaling.

PTP1B inhibition suggests a therapeutic strategy for Rett syndrome

The X-linked neurological disorder Rett syndrome (RTT) presents with autistic features and is caused primarily by mutations in a transcriptional regulator, methyl CpG-binding protein 2 (MECP2). Current treatment options for RTT are limited to alleviating some neurological symptoms; hence, more effective therapeutic strategies are needed. We identified the protein tyrosine phosphatase PTP1B as a therapeutic candidate for treatment of RTT. We demonstrated that the PTPN1 gene, which encodes PTP1B, was a target of MECP2 and that disruption of MECP2 function was associated with increased levels of PTP1B in RTT models. Pharmacological inhibition of PTP1B ameliorated the effects of MECP2 disruption in mouse models of RTT, including improved survival in young male (Mecp2-/y) mice and improved behavior in female heterozygous (Mecp2-/+) mice. We demonstrated that PTP1B was a negative regulator of tyrosine phosphorylation of the tyrosine kinase TRKB, the receptor for brain-derived neurotrophic factor (BDNF). Therefore, the elevated PTP1B that accompanies disruption of MECP2 function in RTT represents a barrier to BDNF signaling. Inhibition of PTP1B led to increased tyrosine phosphorylation of TRKB in the brain, which would augment BDNF signaling. This study presents PTP1B as a mechanism-based therapeutic target for RTT, validating a unique strategy for treating the disease by modifying signal transduction pathways with small-molecule drugs.

The anti-inflammatory compound BAY 11-7082 is a potent inhibitor of Protein Tyrosine Phosphatases

The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small‐molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signaling events, but also may have therapeutic implications. BAY‐11‐7082 is an anti‐inflammatory compound that has been reported to inhibit IκB kinase activity. The compound has an α,β‐unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine‐containing proteins, such as PTPs. In this study, we demonstrated that BAY‐11‐7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY‐11‐7082 inactivated PTPs by forming a covalent adduct with the active‐site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY‐11‐7082 is an effective pan‐PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY‐11‐7082 that have been reported to date.

A Novel Phosphatidic Acid-Protein Tyrosine Phosphatase D2 Axis is Essential for ERBB2 Signaling in Mammary Epithelial Cells

Background: The role of protein-tyrosine phosphatases (PTP) in ERBB2 signaling is undefined. Results: Phosphatidic acid (PA) activated PTPD2; inhibition of PA production or PTPD2 expression attenuated ERBB2-mediated morphological changes in mammary epithelial cells. Conclusion: The PLD2-PTPD2 axis is required for ERBB2 signaling. Significance: PA-regulated PTPD2 activity is a novel, positive element of ERBB2 signaling, which may offer a new therapeutic strategy in breast cancer. We used a loss-of-function screen to investigate the role of classical protein-tyrosine phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling. The study revealed a novel role for PTPD2 as a positive regulator of ERBB2 signaling. Suppression of PTPD2 attenuated the ERBB2-induced multiacinar phenotype in three-dimensional cultures specifically by inhibiting ERBB2-mediated loss of polarity and lumen filling. In contrast, overexpression of PTPD2 enhanced the ERBB2 phenotype. We also found that a lipid second messenger, phosphatidic acid, bound PTPD2 in vitro and enhanced its catalytic activity. Small molecule inhibitors of phospholipase D (PLD), an enzyme that produces phosphatidic acid in cells, also attenuated the ERBB2 phenotype. Exogenously added phosphatidic acid rescued the PLD-inhibition phenotype, but only when PTPD2 was present. These findings illustrate a novel pathway involving PTPD2 and the lipid second messenger phosphatidic acid that promotes ERBB2 function.

A potent, selective and orally bioavailable inhibitor of the protein tyrosine phosphatase PTP1B improves insulin and leptin signaling in animal models

The protein-tyrosine phosphatase PTP1B is a negative regulator of insulin and leptin signaling and a highly validated therapeutic target for diabetes and obesity. Conventional approaches to drug development have produced potent and specific PTP1B inhibitors, but these inhibitors lack oral bioavailability, which limits their potential for drug development. Here, we report that DPM-1001, an analog of the specific PTP1B inhibitor trodusquemine (MSI-1436), is a potent, specific, and orally bioavailable inhibitor of PTP1B. DPM-1001 also chelates copper, which enhanced its potency as a PTP1B inhibitor. DPM-1001 displayed anti-diabetic properties that were associated with enhanced signaling through insulin and leptin receptors in animal models of diet-induced obesity. Therefore, DPM-1001 represents a proof of concept for a new approach to therapeutic intervention in diabetes and obesity. Although the PTPs have been considered undruggable, the findings of this study suggest that allosteric PTP inhibitors may help reinvigorate drug development efforts that focus on this important family of signal-transducing enzymes.

Anxious moments for the protein tyrosine phosphatase PTP1B

Chronic stress can lead to the development of anxiety and mood disorders. Thus, novel therapies for preventing adverse effects of stress are vitally important. Recently, the protein tyrosine phosphatase PTP1B was identified as a novel regulator of stress-induced anxiety. This opens up exciting opportunities to exploit PTP1B inhibitors as anxiolytics.

DPM-1001 decreased copper levels and ameliorated deficits in a mouse model of Wilson's disease

The levels of copper, which is an essential element in living organisms, are under tight homeostatic control. Inactivating mutations in ATP7B, a P-type Cu-ATPase that functions in copper excretion, promote aberrant accumulation of the metal, primarily the in liver and brain. This condition underlies Wilsons disease, a severe autosomal recessive disorder characterized by profound hepatic and neurological deficits. Current treatment regimens rely on the use of broad specificity metal chelators as decoppering agents; however, there are side effects that limit their effectiveness. Here, we present the characterization of DPM-1001 {methyl 4-[7-hydroxy-10,13-dimethyl-3-({4-[(pyridin-2-ylmethyl)amino]butyl}amino)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl] pentanoate} as a potent and highly selective chelator of copper that is orally bioavailable. Treatment of cell models, including fibroblasts derived from Wilsons disease patients, eliminated adverse effects associated with copper accumulation. Furthermore, treatment of the toxic milk mouse model of Wilsons disease with DPM-1001 lowered the levels of copper in the liver and brain, removing excess copper by excretion in the feces while ameliorating symptoms associated with the disease. These data suggest that it may be worthwhile to investigate DPM-1001 further as a new therapeutic agent for the treatment of Wilsons disease, with potential for application in other indications associated with elevated copper, including cancer and neurodegenerative diseases.