Yantao He

Cutting Edge: The PTPN22 Allelic Variant Associated with Autoimmunity Impairs B Cell Signaling

PTPN22 is a gene encoding the protein tyrosine phosphatase Lyp. A missense mutation changing residue 1858 from cytosine to thymidine (1858C/T) is associated with multiple autoimmune disorders. Studies have demonstrated that Lyp has an inhibitory effect on TCR signaling; however, the presence of autoantibodies in all of the diseases associated with the 1858T variant and recent evidence that Ca2+ flux is altered in B cells of 1858T carriers indicate a role for Lyp in B cell signaling. In this study we show that B cell signal transduction is impaired in individuals who express the variant. This defect in signaling is characterized by a deficit in proliferation, a decrease in phosphorylation of key signaling proteins, and is reversed by inhibition of Lyp. These findings suggest that the PTPN22 1858T variant alters BCR signaling and implicate B cells in the mechanism by which the PTPN22 1858T variant contributes to autoimmunity.

Targeting mycobacterium protein tyrosine phosphatase B for antituberculosis agents

Protein tyrosine phosphatases are often exploited and subverted by pathogenic bacteria to cause human diseases. The tyrosine phosphatase mPTPB from Mycobacterium tuberculosis is an essential virulence factor that is secreted by the bacterium into the cytoplasm of macrophages, where it mediates mycobacterial survival in the host. Consequently, there is considerable interest in understanding the mechanism by which mPTPB evades the host immune responses, and in developing potent and selective mPTPB inhibitors as unique antituberculosis (antiTB) agents. We uncovered that mPTPB subverts the innate immune responses by blocking the ERK1/2 and p38 mediated IL-6 production and promoting host cell survival by activating the Akt pathway. We identified a potent and selective mPTPB inhibitor I-A09 with highly efficacious cellular activity, from a combinatorial library of bidentate benzofuran salicylic acid derivatives assembled by click chemistry. We demonstrated that inhibition of mPTPB with I-A09 in macrophages reverses the altered host immune responses induced by the bacterial phosphatase and prevents TB growth in host cells. The results provide the necessary proof-of-principle data to support the notion that specific inhibitors of the mPTPB may serve as effective antiTB therapeutics.

Structure, inhibitor, and regulatory mechanism of Lyp, a lymphoid-specific tyrosine phosphatase implicated in autoimmune diseases

The lymphoid-specific tyrosine phosphatase (Lyp) has generated enormous interest because a single-nucleotide polymorphism in the gene (PTPN22) encoding Lyp produces a gain-of-function mutant phosphatase that is associated with several autoimmune diseases, including type I diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus. Thus, Lyp represents a potential target for a broad spectrum of autoimmune disorders. Unfortunately, no Lyp inhibitor has been reported. In addition, little is known about the structure and biochemical mechanism that directly regulates Lyp function. Here, we report the identification of a bidentate salicylic acid-based Lyp inhibitor I-C11 with excellent cellular efficacy. Structural and mutational analyses indicate that the inhibitor binds both the active site and a nearby peripheral site unique to Lyp, thereby furnishing a solid foundation upon which inhibitors with therapeutic potency and selectivity can be developed. Moreover, a comparison of the apo- and inhibitor-bound Lyp structures reveals that the Lyp-specific region S35TKYKADK42, which harbors a PKC phosphorylation site, could adopt either a loop or helical conformation. We show that Lyp is phosphorylated exclusively at Ser-35 by PKC both in vitro and in vivo. We provide evidence that the status of Ser-35 phosphorylation may dictate the conformational state of the insert region and thus Lyp substrate recognition. We demonstrate that Ser-35 phosphorylation impairs Lyps ability to inactivate the Src family kinases and down-regulate T cell receptor signaling. Our data establish a mechanism by which PKC could attenuate the cellular function of Lyp, thereby augmenting T cell activation.

Salicylic acid based small molecule inhibitor for the oncogenic Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2).

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

Small molecule tools for functional interrogation of protein tyrosine phosphatases

The importance of protein tyrosine phosphatases (PTPs) in the regulation of cellular signalling is well established. Malfunction of PTP activity is also known to be associated with cancer, metabolic syndromes and autoimmune disorders, as well as neurodegenerative and infectious diseases. However, a detailed understanding of the roles played by the PTPs in normal physiology and in pathogenic conditions has been hampered by the absence of PTP‐specific small molecule agents. In addition, the therapeutic benefits of modulating this target class are underexplored as a result of a lack of suitable chemical probes. Potent and specific PTP inhibitors could significantly facilitate functional analysis of the PTPs in complex cellular signal transduction pathways and may constitute valuable therapeutics in the treatment of several human diseases. We highlight the current challenges to and opportunities for developing PTP‐specific small molecule agents. We also review available selective small molecule inhibitors developed for a number of PTPs, including PTP1B, TC‐PTP, SHP2, lymphoid‐specific tyrosine phosphatase, haematopoietic protein tyrosine phosphatase, CD45, PTPβ, PTPγ, PTPRO, Vaccinia H1‐related phosphatase, mitogen‐activated protein kinase phosphatase‐1, mitogen‐activated protein kinase phosphatase‐3, Cdc25, YopH, mPTPA and mPTPB.

Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

Noonan syndrome (NS) is an autosomal dominant congenital disorder characterized by multiple birth defects including heart defects and myeloproliferative disease (MPD). Approximately 50% of NS patients have germline gain-of-function mutations in PTPN11, which encodes the protein-tyrosine phosphatase, Shp2. We provide evidence that conditional ablation of Stat3 in hematopoietic cells and cardiac valvular tissues leads to myeloid progenitor hyperplasia and pulmonary stenosis due to the leaflet thickening, respectively. Consistently, STAT3 activation is significantly compromised in peripheral blood cells from NS patients bearing Shp2-activating mutations. Biochemical and functional analyses demonstrate that activated Shp2 is able to down-regulate Tyr(P)-Stat3 and that constitutively active Stat3 rescues activating mutant Shp2-induced granulocyte-macrophage colony-stimulating factor hypersensitivity in bone marrow cells. Collectively, our work demonstrates that Stat3 is an essential signaling component potentially contributing to the pathogenesis of NS and juvenile myelomonocytic leukemia caused by PTPN11 gain-of-function mutations.

Discovery and evaluation of novel inhibitors of mycobacterium protein tyrosine phosphatase B from the 6-Hydroxy-benzofuran-5-carboxylic acid scaffold.

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (mPTPB) is a virulence factor secreted by the pathogen and mediates mycobacterial survival in macrophages by targeting host cell immune responses. Consequently, mPTPB represents an exciting new target to combat tuberculosis (TB) infection. We describe a medicinal chemistry oriented approach that transforms a benzofuran salicylic acid scaffold into a highly potent (IC(50) = 38 nM) and selective mPTPB inhibitor (>50 fold against a large panel of PTPs). Importantly, the inhibitor is capable of reversing the altered host immune responses induced by the bacterial phosphatase and restoring the macrophages full capacity to secrete IL-6 and undergo apoptosis in response to interferon-γ stimulation, validating the concept that chemical inhibition of mPTPB may be therapeutically useful for novel TB treatment. The study further demonstrates that bicyclic salicylic acid pharmacophores can be used to deliver PTP inhibitors with high potency, selectivity, and cellular efficacy.

Negative regulation of TLR signaling in myeloid cells--implications for autoimmune diseases.

Toll‐like receptors (TLR) are transmembrane pattern recognition receptors that recognize microbial ligands and signal for production of inflammatory cytokines and type I interferon in macrophages and dendritic cells (DC). Whereas TLR‐induced inflammatory mediators are required for pathogen clearance, many are toxic to the host and can cause pathological inflammation when over‐produced. This is demonstrated by the role of TLR‐induced cytokines in autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. Because of the potent effects of TLR‐induced cytokines, we have diverse mechanisms to dampen TLR signaling. Here, we highlight three pathways that participate in inhibition of TLR responses in macrophages and DC, and their implications in autoimmunity; A20, encoded by the TNFAIP3 gene, Lyp encoded by the PTPN22 gene, and the BCAP/PI3K pathway. We present new findings that Lyp promotes TLR responses in primary human monocytes and that the autoimmunity risk Lyp620W variant is more effective at promoting TLR‐induced interleukin‐6 than the non‐risk Lyp620R protein. This suggests that Lyp serves to downregulate a TLR inhibitory pathway in monocytes, and we propose that Lyp inhibits the TREM2/DAP12 inhibitory pathway. Overall, these pathways demonstrate distinct mechanisms of negative regulation of TLR responses, and all impact autoimmune disease pathogenesis and treatment.

The protein tyrosine phosphatase, Shp2, positively contributes to FLT3-ITD-induced hematopoietic progenitor hyperproliferation and malignant disease in vivo

Internal tandem duplications (ITDs) in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia (AML). We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knockdown of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low-density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML.