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

Orrù, Valeria; Tsai, Sophia J.; Rueda, Blanca; Fiorillo, Edoardo; Stanford, Stephanie M.; Dasgupta, J.; Hartiala, Jaana; Zhao, Lei; Ortego-Centeno, Norbérto; D’Alfonso, Sandra; Arnett, Frank C.; Wu, Hui; González-Gay, Miguel Á.; Tsao, B. P.; Pons-Éstel, Bernardo A; Alarcón‐Riquelme, Marta E.; He, Yantao; Zhang, Z.-Y.; Allayee, Hooman; Chen, X. S.; Martı́n, Javier; Bottini, Nunzio

doi:10.1093/hmg/ddn363

Cited by 112 publications

(127 citation statements)

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“…First, because the charge and hydrophobicity of surfaces surrounding the catalytic pocket vary considerably among PTPs (15,(18)(19)(20), we explored whether the addition of the polyarginine tag with a lipid tail restricts the selectivity of the peptides. A cell-penetrating version of peptide 1 (myristoyl-βAla-(Arg) 7 -EDNE-(pCAP)-TARE-NH 2 ; hereafter, SP1) was synthesized.…”

Section: Resultsmentioning

confidence: 99%

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45

Stanford

Panchal

Walker

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157-177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107-137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors.single-cell assay | peptide substrate P rotein tyrosine phosphatases (PTPs) are important regulators of signal transduction and are emerging as drug targets for common and debilitating human diseases, including cancer, diabetes, arthritis, and infectious diseases (1). The transmembrane PTP CD45, a critical regulator of signaling through the T-cell receptor (TCR), was one of the first PTPs to be considered as a drug target for treatment of human autoimmune diseases (2). In addition, inhibitors of PTP-1B and SHP-2 currently are being sought for therapy of metabolic diseases and cancer, respectively (3-5). Unfortunately, development of small-molecule inhibitors of PTPs has been a challenging task. Poor cell permeability of PTP inhibitors has been a limitation difficult to overcome in later stages of drug development (5). Several methods to assess intracellular PTP activity are available (6-8), yet no assay has been able to capture intracellular PTP activity at the single-cell level. Cell-based PTP assays that work at the single-cell level and are suited to high-throughput screening might accelerate PTP inhibitor development.In this report we describe the development and optimization of a method to monitor PTP activity at the single-cell level by using cell-permeable fluorogenic peptides that, once delivered into cells, are dephosphorylated by PTPs. The dephosphorylation of these peptides by PTPs generates a signal that can be monitored by flow cytometry and h...

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Section: Resultsmentioning

confidence: 99%

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45

Stanford

Panchal

Walker

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…31 Another less frequent PTPN22 variant (R263Q), which has been associated with reduced risk for autoimmune disease, was observed in another patient. 32 Overall, the sequencing analysis revealed that PTPN22 and SHP-1 are not frequently mutated in CLL and that the PTPN22 autoimmunedisease risk variant is not more prevalent in CLL patients than in the general population. …”

mentioning

confidence: 92%

Overexpression of the autoimmunity-associated phosphatase PTPN22 promotes survival of antigen-stimulated CLL cells by selectively activating AKT

Negro

Gobessi

Longo

et al. 2012

Blood

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A polymorphic variant of the phosphatase PTPN22 has been associated with increased risk for multiple autoimmune diseases. The risk allele is thought to function by diminishing antigen-receptor signals responsible for negative selection of autoreactive lymphocytes. We now show that PTPN22 is markedly overexpressed in chronic lymphocytic leukemia (CLL), a common malignancy of autoreactive B lymphocytes. We also show that overexpression of PTPN22 significantly inhibits antigen-induced apoptosis of primary CLL cells by blocking B-cell receptor (BCR) signaling pathways that negatively regulate lymphocyte survival. More importantly, we show that PTPN22 positively regulates the antiapoptotic AKT kinase, which provides a powerful survival signal to antigen-stimulated CLL cells. This selective uncoupling of AKT from other downstream BCR signaling pathways is a result of inhibition of a negative regulatory circuit involving LYN, CD22, and SHIP. Finally, we show that PTPN22 can be effectively down-regulated by the PKC inhibitors ruboxistaurin and sotrastaurin, resulting in enhanced killing of CLL cells exposed to proapoptotic BCR stimuli. Collectively, these data suggest that PTPN22 overexpression represents a protective mechanism that allows autoantigen-activated CLL cells to escape from negative selection and indicate that this mechanism could be exploited for therapeutic purposes by targeting PTPN22 with PKC inhibitors. (Blood. 2012; 119(26):6278-6287) IntroductionChronic lymphocytic leukemia (CLL) is a common lymphoid malignancy characterized by the expansion and progressive accumulation of mature B lymphocytes that coexpress the T-cell antigen CD5 and B cell surface antigens CD19, CD20, and CD23. The disease has a highly variable clinical course, ranging from rapid progression with fatal outcome to a relatively indolent behavior with normal life expectancy. 1 Several lines of evidence suggest that chronic antigen drive plays an important role in the pathogenesis of CLL. 1,2 First, the malignant B cells from different patients frequently express similar or identical B-cell receptors (BCRs), suggesting that they recognize the same antigens and that these antigens drive the initial expansions of the malignant clones. 3 Second, freshly isolated CLL cells show increased expression of BCR target genes and reduced expression of surface IgM, indicating that they are continuously triggered by antigen in vivo. [4][5][6] Third, there is a strong correlation between clinical course and certain BCR-related features, such as the mutational status of the immunoglobulin heavy-chain variable (IGHV) genes and ZAP-70 expression, suggesting that BCR signals also play a role during disease progression. [7][8][9] Lastly, early clinical trials with agents that target the BCR signaling pathway, such as inhibitors of SYK, BTK, and PI3K␦, are showing considerable activity in patients with CLL, further suggesting that the leukemic cells rely on BCR signals for growth and survival. [10][11][12] Despite all this evidence, the malignant B cells al...

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“…Importantly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, leading to increased inhibition of T-cell signaling relative to the wild-type enzyme (12). Interestingly, a loss-of-function variant of PTPN22 has been linked to reduced risk of systemic lupus erythematosus (13). Hence, Lyp is emerging as a potential target for therapeutic intervention of a broad spectrum of autoimmune disorders.…”

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confidence: 99%

Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

Yu¹,

Chen²,

Zhang

et al. 2011

Journal of Biological Chemistry

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A missense single-nucleotide polymorphism in the gene encoding the lymphoid-specific tyrosine phosphatase (Lyp) has been identified as a causal factor in a wide spectrum of autoimmune diseases. Interestingly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, implicating Lyp as an attractive target for the development of effective strategies for the treatment of many autoimmune disorders. Unfortunately, the precise biological functions of Lyp in signaling cascades and cellular physiology are poorly understood. Identification and characterization of Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we identified consensus sequence motifs for Lyp substrate recognition using an "inverse alanine scanning" combinatorial library approach. The intrinsic sequence specificity data led to the discovery and characterization of SKAP-HOM, a cytosolic adaptor protein required for proper activation of the immune system, as a bona fide Lyp substrate. To determine the molecular basis for Lyp substrate recognition, we solved crystal structures of Lyp in complex with the consensus peptide as well as the phosphopeptide derived from SKAP-HOM. Together with the biochemical data, the structures define the molecular determinants for Lyp substrate specificity and provide a solid foundation upon which novel therapeutics targeting Lyp can be developed for multiple autoimmune diseases.Protein-tyrosine phosphorylation-mediated signal transduction is essential for a wide range of eukaryotic functions, including cell proliferation, differentiation, migration, apoptosis, and the immune responses (1). This signaling mechanism is often dysregulated in human diseases, due to aberrant activity of the enzymes involved in the regulation of protein tyrosine phosphorylation status (1, 2). Thus, a comprehensive understanding of the physiological roles of protein tyrosine phosphorylation and how this process is abrogated in the pathogenesis of human diseases must necessarily include characterization of proteintyrosine phosphatases (PTPs), 4 in addition to protein-tyrosine kinases.The lymphoid-specific tyrosine phosphatase (Lyp) has garnered tremendous interest due to the observation that a missense C1858T single nucleotide polymorphism in the gene (PTPN22) encoding Lyp is associated with multiple autoimmune disorders, including type I diabetes (3), rheumatoid arthritis (4, 5), Graves disease (6), and systemic lupus erythematosus (7). Lyp expression is restricted to human T cells, B cells, and macrophages (8). Lyp exerts an inhibitory effect on the proximal T cell receptor signaling pathways, probably through dephosphorylation of the Lck and ZAP-70 kinases (9 -11). The C1858T single nucleotide polymorphism changes Arg 620 into a Trp within the first Pro-rich region in the C terminus of Lyp, leading to loss of Lyp binding to the Src homology 3 domain of the Src C-terminal kinase (Csk) (3, 4). Importantly, the autoimmune-predisposing variant of...

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

Cited by 112 publications

References 29 publications

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45

Overexpression of the autoimmunity-associated phosphatase PTPN22 promotes survival of antigen-stimulated CLL cells by selectively activating AKT

Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

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