T-cell Protein Tyrosine Phosphatase Research Articles

Site-Specifically Modified Peptide Inhibitors of Protein Tyrosine Phosphatase 1B and T-Cell Protein Tyrosine Phosphatase with Enhanced Stability and Improved In Vivo Long-Acting Activity.

Protein tyrosine phosphatase 1B (PTP1B) and TC-PTP can function in a coordinated manner to regulate diverse biological processes including insulin and leptin signaling, T-cell activation, and tumor antigen presentation, which makes them potential targets for several therapeutic applications. We have previously demonstrated that the lipidated BimBH3 peptide analogues were a new class of promising PTP1B inhibitors with once-weekly antidiabetic potency. Herein, we chemically synthesized two series of BimBH3 analogues via site-specific modification and studied their structure-activity relationship. The screened analogues S2, S6, A2-14, A2-17, A2-20, and A2-21 exhibited an improved PTP1B/TC-PTP dual inhibitory activity and achieved good stability in the plasma of mice and dogs, which indicated long-acting potential. In mouse models of type 2 diabetes mellitus (T2DM), the selected analogues S6, S7, A2-20, and A2-21 with an excellent target activity and plasma stability generated once-weekly therapeutic potency for T2DM at lower dosage (0.5 μmol/kg). In addition, evidence was provided to confirm the cell permeability and targeted enrichment of the BimBH3 analogues. In summary, we report here that site-specific modification and long fatty acid conjugation afforded cell-permeable peptidomimetic analogues of BimBH3 with enhanced stability, in vivo activity, and long-acting pharmacokinetic profile. Our findings could guide the further optimization of BimBH3 analogues and provide a proof-of-concept for PTP1B/TC-PTP targeting as a new therapeutic approach for T2DM, which may facilitate the discovery and development of alternative once-weekly anti-T2DM drug candidates.

Chemical Epigenetic Regulation Secondary Metabolites Derived from Aspergillus sydowii DL1045 with Inhibitory Activities for Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are ubiquitous in living organisms and are promising drug targets for cancer, diabetes/obesity, and autoimmune disorders. In this study, a histone deacetylase inhibitor called suberoylanilide hydroxamic acid (SAHA) was added to a culture of marine fungi (Aspergillus sydowii DL1045) to identify potential drug candidates related to PTP inhibition. Then, the profile of the induced metabolites was characterized using an integrated metabolomics strategy. In total, 46% of the total SMs were regulated secondary metabolites (SMs), among which 20 newly biosynthesized metabolites (10% of the total SMs) were identified only in chemical epigenetic regulation (CER) broth. One was identified as a novel compound, and fourteen compounds were identified from Aspergillus sydowii first. SAHA derivatives were also biotransformed by A. sydowii DL1045, and five of these derivatives were identified. Based on the bioassay, some of the newly synthesized metabolites exhibited inhibitory effects on PTPs. The novel compound sydowimide A (A11) inhibited Src homology region 2 domain-containing phosphatase-1 (SHP1), T-cell protein tyrosine phosphatase (TCPTP) and leukocyte common antigen (CD45), with IC50 values of 1.5, 2.4 and 18.83 μM, respectively. Diorcinol (A3) displayed the strongest inhibitory effect on SHP1, with an IC50 value of 0.96 μM. The structure-activity relationship analysis and docking studies of A3 analogs indicated that the substitution of the carboxyl group reduced the activity of A3. Research has demonstrated that CER positively impacts changes in the secondary metabolic patterns of A. sydowii DL1045. The compounds produced through this approach will provide valuable insights for the creation and advancement of novel drug candidates related to PTP inhibition.

Immunotherapeutic implications of negative regulation by protein tyrosine phosphatases in T cells: the emerging cases of PTP1B and TCPTP.

In the context of inflammation, T cell activation occurs by the concerted signals of the T cell receptor (TCR), co-stimulatory receptors ligation, and a pro-inflammatory cytokine microenvironment. Fine-tuning these signals is crucial to maintain T cell homeostasis and prevent self-reactivity while offering protection against infectious diseases and cancer. Recent developments in understanding the complex crosstalk between the molecular events controlling T cell activation and the balancing regulatory cues offer novel approaches for the development of T cell-based immunotherapies. Among the complex regulatory processes, the balance between protein tyrosine kinases (PTK) and the protein tyrosine phosphatases (PTPs) controls the transcriptional and metabolic programs that determine T cell function, fate decision, and activation. In those, PTPs are de facto regulators of signaling in T cells acting for the most part as negative regulators of the canonical TCR pathway, costimulatory molecules such as CD28, and cytokine signaling. In this review, we examine the function of two close PTP homologs, PTP1B (PTPN1) and T-cell PTP (TCPTP; PTPN2), which have been recently identified as promising candidates for novel T-cell immunotherapeutic approaches. Herein, we focus on recent studies that examine the known contributions of these PTPs to T-cell development, homeostasis, and T-cell-mediated immunity. Additionally, we describe the signaling networks that underscored the ability of TCPTP and PTP1B, either individually and notably in combination, to attenuate TCR and JAK/STAT signals affecting T cell responses. Thus, we anticipate that uncovering the role of these two PTPs in T-cell biology may lead to new treatment strategies in the field of cancer immunotherapy. This review concludes by exploring the impacts and risks that pharmacological inhibition of these PTP enzymes offers as a therapeutic approach in T-cell-based immunotherapies.

Biophysical Rationale for the Selective Inhibition of PTP1B over TCPTP by Nonpolar Terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L-11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a β site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the β site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).

Cannabinoid receptor type 1 (CB1R) inhibits hypothalamic leptin signaling via β-arrestin1 in complex with TC-PTP and STAT3

Molecular interactions between anorexigenic leptin and orexigenic endocannabinoids, although of great metabolic significance, are not well understood. Wereport here that hypothalamic STAT3 signaling in mice, initiated by physiological elevations of leptin, is diminished by agonists of the cannabinoid receptor 1 (CB1R). Measurement of STAT3 activation by semi-automated confocal microscopy in cultured neurons revealed that this CB1R-mediated inhibition requires both Tcell protein tyrosine phosphatase (TC-PTP) and β-arrestin1 but is independent of changes in cAMP. Moreover, β-arrestin1 translocates to the nucleus upon CB1R activation and binds both STAT3 and TC-PTP. Consistently, CB1R activation failed to suppress leptin signaling in β-arrestin1 knockout mice invivo, and in neural cells deficient in CB1R, β-arrestin1 or TC-PTP. Altogether, CB1R activation engages β-arrestin1 to coordinate the TC-PTP-mediated inhibition of the leptin-evoked neuronal STAT3 response. This mechanism may restrict the anorexigenic effects of leptin when hypothalamic endocannabinoid levels rise, as during fasting or in diet-induced obesity.

The IBD-candidate gene, PTPN2, is a Key Regulator of Ileal Paneth Cell Function in Mice

Loss-of-function variants in the Protein Tyrosine Phosphatase non-receptor Type 2 ( PTPN2) gene are associated with increased risk of Inflammatory Bowel Disease (IBD). PTPN2 encodes T Cell Protein Tyrosine Phosphatase (TCPTP), a negative regulator of intracellular signaling pathways including JAK-STAT. PTPN2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage crosstalk, and modulating the gut microbiome in mice, although the mechanism in which PTPN2 loss affects gut microbiome is unknown. IBD patient biopsies and studies in transgenic mice show that IBD-associated genes can modify IEC subtypes and their functional roles in intestinal homeostasis. Hypothesis: PTPN2 loss compromises modulation of gut microbiome by impairing number and function of IEC subtypes. Methods: To identify how loss of PTPN2 activity in vivo affects ileal IEC subtypes and their function, RNA and protein were isolated from ileal IECs of whole-body Ptpn2-wild-type (WT), heterozygous (Het) and KO mice. RNA was assayed by two separate Nanostring ® panels: PanCancer and AutoImmune totaling >1500 targets. Dysregulated targets associated with IEC function were validated by Western blot, immunostaining, and transmission electron microscopy (TEM). Altered genes were then investigated in tamoxifen-inducible intestinal epithelial-specific Ptpn2 knockout mice ( Ptpn2 Δ IEC ) and Ptpn2 fl/fl controls. Results: 1398 targets had detectable expression. Notably, Paneth cell (PC)-associated genes Lyz1, Defa6, Pla2g2a, and Fgfr3 were markedly downregulated (p<0.05 and FDR<0.1; n=4). Reduced PC numbers in whole-body Ptpn2-KO mice were confirmed by decreased Lysozyme and Cryptdin-1 staining (p<0.001; n=5), reduced Lysozyme protein levels by western blotting (p=0.004; n=5), and loss of PC phenotype by TEM (n=3). Ki-67 staining revealed elevated ileal crypt cell proliferation (p=0.02; n=3) and longer villi (p=0.03; n=9) in Ptpn2-KO mice vs. WT. However, unchanged cleaved caspase 3 and TUNEL staining indicated no increase in IEC apoptosis. Further, TEM analysis showed disrupted endoplasmic reticulum (ER) architecture in PCs of Ptpn2-KO mice (n=3) accompanied by higher protein levels of the ER stress marker CHOP (p=0.01; n=8). Conversely, Ptpn2 Δ IEC mice showed no change in ileal villus length (n=6), number of PCs (n=7), and TEM showed abundant cytosolic granules compared to Ptpn2 fl/fl (n=4). However, immunostaining (n=6) and Western blotting (p<0.05; n=6) showed reduced Lysozyme protein accompanied by elevated levels of unfolded protein response Xbp-1s in Ptpn2 Δ IEC mice (p<0.05; n=6). Conclusion: Ptpn2-deficiency affects Paneth cell viability by decreasing cell number, promoting ER stress, and impairing production of PC-associated antimicrobial peptides. This likely contributes to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2-deficient mice and may explain the association of PTPN2 variants with dysbiosis in clinical IBD. Supported by NIH 2R01DK091281, 1R01AI153314-01, R21AI152017 and R01AI165490 (DFM) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The IBD-candidate gene, PTPN2, Regulates Goblet Cell Number and Expression of Intestinal Epithelial Cell Markers in a Regional-specific Manner in Mice

Loss-of-function variants in the Protein Tyrosine Phosphatase non-receptor Type 2 ( PTPN2) gene are associated with increased risk of Inflammatory Bowel Disease (IBD). PTPN2 encodes T Cell Protein Tyrosine Phosphatase (TCPTP), a negative regulator of intracellular signaling pathways including JAK-STAT. PTPN2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage crosstalk, and modulating the gut microbiota in mice and IBD patients. Aim: To determine if Ptpn2 loss compromises IEC subtypes that restrict gut dysbiosis. Methods: Ileal and colonic IEC subtypes and their function were assessed by PAS staining of goblet cells and immunostaining. RNA isolated from colonic IECs of whole-body Ptpn2-wild-type (WT), heterozygous (Het) and KO mice was assayed by two separate Nanostring ® panels: PanCancer and AutoImmune totaling >1500 targets. Flow cytometry was performed to evaluate abundance of mucosal-associated immune cells. Results: Goblet cells were reduced in the ileal and cecal crypts compared with WT mice (p<0.05; n=5) while numbers in the proximal and distal colon were variable. Ki-67 immunostaining revealed increased proliferating cells in the proximal and distal colon (p<0.01; n=3). Nanostring analysis had 1399 targets with detectable expression. Notably, expression of growth factors Egf and Fgfr3, differentiation factors Atoh1 and Mycn, tuft cell marker Dclk1, and intestinal stem cell marker Lgr5, were all downregulated in colonic IECs from Ptpn2-KO mice compared with WT (p<0.05; FDR<0.1; n=4). Moreover, abundance of mucosal neutrophils, macrophages, CD4 + expressing IFN-γ, CD8 + expressing TNF/IFN-γ, and CD4 + expressing IL-22 T-cells were higher in colon and cecum of Ptpn2-KO mice compared with WT and HET mice (p<0.01; n=5), whereas abundance of IL-4 expressing CD4 + cells, and Tregs expressing IL-10 were decreased in all intestinal segments of Ptpn2-KO mice (p<0.01; n=5). Conclusion: Ptpn2 loss negatively impacts abundance of IEC subtypes in a region-specific manner, affecting epithelial proliferation and expression of critical differentiation/cell markers. The elevated abundance of immune cells expressing pro-inflammatory signals accompanied by decreased levels of cells expressing IL-4 and IL-10, could contribute to a skewed abundance of IEC subtypes and their function. The observed effects may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2-deficient mice, and partly explain association of PTPN2 variants with dysbiosis in clinical IBD. Supported by NIH 2R01DK091281, 1R01AI153314-01, R21AI152017 and R01AI165490 (DFM). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 625: Alteration Of Circulating Ace2-network Related Micrornas In Patients With Covid-19

ACE2 interaction network caused by virus infection can affect the expression levels of miRNAs and influence severity of COVID19. This study aimed to analyse the diagnostic/predictive utility of ACE2-related miRNAs, identified by in silico analysis: miR10b, miR124, miR200b-3p, miR26b, miR302c-5p in patients with COVID-19, and aimed to unravel the functions of miRNAs, by using machine learning-SHAP analysis for clinical data and bioinformatic tools. Blood samples and clinical data of 79 patients and 32 healthy were collected at; day of admission, 7days and 21days after admission. Endpoint was hospitalisation length of stay (>21days) and/or death in follow-up. Delta low miR200b-3p expression (7days-admission) presents predictive utility in assessment of the hospital length of stay and/or death (AUC:0.73,p=0.002). Logistic regression analysis showed that delta low miR-200b-3p, diabetes are independent predictors of increased hospital length of stay and/or death (OR=5.8;CI=0.57-21.21;p= 0.008, OR= 4.9;CI=1-23.9;p=0.04). MiR-26b-5p and miR10b in patients were found lower at the baseline, 7 and 21days after admission compared to healthy controls (p<0.0001 for all time points). SHAP analysis showed miR200b-3p (day7), miR302c-5p (day7), CRP (day7), neutrophils (day0), and DDimer (day0) as most promising predictors of long hospitalisation. Pathway-enrichment showed the following top pathways of the interleukin-2 signaling pathway, and cancer pathways. MiR200b-3p showed regulation of COVID19-related targets associated with Tcell protein tyrosine phosphatase and HIF-1 transcriptional activity in hypoxia, key pathways in COVID19. Bioinformatics analysis showed miRNAs roles in multiple CVDs phenotypes associated with COVID19. We validated/characterized miRNAs which can serve as novel, predictive biomarkers of the long term COVID19 hospitalisation and can be used for early stratification of patients and prediction of severity of infection.

Small Molecule Degraders of Protein Tyrosine Phosphatase 1B and T‐Cell Protein Tyrosine Phosphatase for Cancer Immunotherapy

AbstractProtein tyrosine phosphatase 1B (PTP1B) and T‐cell protein tyrosine phosphatase (TC‐PTP) play non‐redundant negative regulatory roles in T‐cell activation, tumor antigen presentation, insulin and leptin signaling, and are potential targets for several therapeutic applications. Here, we report the development of a highly potent and selective small molecule degrader DU‐14 for both PTP1B and TC‐PTP. DU‐14 mediated PTP1B and TC‐PTP degradation requires both target protein(s) and VHL E3 ligase engagement and is also ubiquitination‐ and proteasome‐dependent. DU‐14 enhances IFN‐γ induced JAK1/2‐STAT1 pathway activation and promotes MHC‐I expression in tumor cells. DU‐14 also activates CD8+ T‐cells and augments STAT1 and STAT5 phosphorylation. Importantly, DU‐14 induces PTP1B and TC‐PTP degradation in vivo and suppresses MC38 syngeneic tumor growth. The results indicate that DU‐14, as the first PTP1B and TC‐PTP dual degrader, merits further development for treating cancer and other indications.

Small Molecule Degraders of Protein Tyrosine Phosphatase 1B and T-Cell Protein Tyrosine Phosphatase for Cancer Immunotherapy.

Protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TC-PTP) play non-redundant negative regulatory roles in T-cell activation, tumor antigen presentation, insulin and leptin signaling, and are potential targets for several therapeutic applications. Here, we report the development of a highly potent and selective small molecule degrader DU-14 for both PTP1B and TC-PTP. DU-14 mediated PTP1B and TC-PTP degradation requires both target protein(s) and VHL E3 ligase engagement and is also ubiquitination- and proteasome-dependent. DU-14 enhances IFN-γ induced JAK1/2-STAT1 pathway activation and promotes MHC-I expression in tumor cells. DU-14 also activates CD8+ T-cells and augments STAT1 and STAT5 phosphorylation. Importantly, DU-14 induces PTP1B and TC-PTP degradation in vivo and suppresses MC38 syngeneic tumor growth. The results indicate that DU-14, as the first PTP1B and TC-PTP dual degrader, merits further development for treating cancer and other indications.

Abstract 2558: Loss of TC-PTP in keratinocytes leads to increased UVB-induced autophagy

Abstract Amongst the deteriorating effects of the sun’s ultraviolet B (UVB) radiation on cellular homeostasis is the formation of photoproducts in the DNA, such as cyclobutene pyrimidine dimers. Photoproducts cause conformational alterations in the DNA structure which may evade cellular repair machinery and persist through DNA replication processes, leading to potential gene mutations and ultimately leading to skin cancer. Photoproducts are typically repaired by nucleotide excision repair. Notwithstanding, when the repair mechanism fails, apoptosis ensues to prevent the accumulation of mutations and to restore cellular homeostasis. T-cell protein tyrosine phosphatase (TC-PTP) is one of the members of PTP family that has been shown to increase UVB-induced apoptosis in keratinocytes via the downregulation of Flk-1/JNK signaling. This implies that loss of TC-PTP causes resistance to apoptosis in UVB-damaged keratinocytes. In the present work, we report that this resistance is accompanied by an increase in UVB-induced autophagy in the absence of TC-PTP. We generated human TC-PTP-deficient (TC-PTP/KO) HaCaT keratinocytes using CRISPR/Cas9 system. The expression of microtubule-associated protein light chain 3 (LC3) was significantly increased in TC-PTP/KO HaCaT keratinocytes compared to engineered controls (TC-PTP/Mock) following treatment with UVB irradiation. Increased expression of LC3 in TC-PTP/KO keratinocytes was accompanied by a significant decrease in the expression of the p62. We also observed that treatment of TC-PTP KO keratinocytes with chloroquine (CQ), a late-phase autophagy inhibitor, caused a remarkable increase in LC3 expression following UVB exposure in comparison with untreated TC-PTP/KO keratinocytes, implying that loss of TC-PTP increases autophagy in response to UVB. With this regard, TC-PTP deficiency in human keratinocytes significantly increased cell proliferation following UVB exposure compared to similarly treated control keratinocytes. Pretreatment of CQ before UVB irradiation in TC-PTP KO keratinocytes significantly reduced cell viability compared to untreated TC-PTP KO keratinocytes. These results suggest that TC-PTP protects keratinocytes from UVB-induced damage by negatively regulating autophagy signaling. Citation Format: Obed Asare, Klarissa Zavala, Lindsey Shim, Bilal Hafeez, Dae Kim. Loss of TC-PTP in keratinocytes leads to increased UVB-induced autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2558.

Isosteviol derivatives as protein tyrosine Phosphatase-1B inhibitors: Synthesis, biological evaluation and molecular docking.

Protein tyrosine phosphatase (PTP1B) antagonizes insulin signaling and acts as a potential therapeutic target for insulin resistance associated with obesity and type II diabetes. In this work, a series of isosteviol derivatives 1-28 was synthesized and the inhibitory activity on PTP1B was evaluated by double antibody sandwich ELISA (DAS-ELISA) in vitro. Most isosteviol derivatives showed moderate PTP1B inhibitory activities. Among them, derivatives 10, 13, 24, 27 showed remarkable bioactivities with IC50 values ranging from 0.24 to 0.40µM. Particularly, derivative 24 exhibited the best inhibitory activity against PTP1B (IC50=0.24µM) in vitro; moreover, it showed 7-fold selectivity to PTP1B over T-cell protein tyrosine phosphatase (TCPTP) and 14-fold selectivity to PTP1B over cell division cycle 25 homolog B (CDC25B). Molecular docking studies demonstrated the hydrogen bond interaction between 24 and LYS-116 residue in PTP1B might be essential for the inhibitory activity. The results suggested that derivative 24 has great potential to be employed as drug candidate for the treatment of obesity and type II diabetes.

Targeting protein tyrosine phosphatases for CDK6-induced immunotherapy resistance

Elucidating the mechanisms of resistance to immunotherapy and developing strategies to improve its efficacy are challenging goals. Bioinformatics analysis demonstrates that high CDK6 expression in melanoma is associated with poor progression-free survival of patients receiving single-agent immunotherapy. Depletion of CDK6 or cyclin D3 (but not of CDK4, cyclin D1, or D2) in cells of the tumor microenvironment inhibits tumor growth. CDK6 depletion reshapes the tumor immune microenvironment, and the host anti-tumor effect depends on cyclin D3/CDK6-expressing CD8+ and CD4+ Tcells. This occurs by CDK6 phosphorylating and increasing the activities of PTP1B and Tcell protein tyrosine phosphatase (TCPTP), which, in turn, decreases tyrosine phosphorylation of CD3ζ, reducing the signal transduction for Tcell activation. Administration of a PTP1B and TCPTP inhibitor prove more efficacious than using a CDK6 degrader in enhancing Tcell-mediated immunotherapy. Targeting protein tyrosine phosphatases (PTPs) might be an effective strategy for cancer patients who resist immunotherapy treatment.

Epigenetic modification of CSDE1 locus dictates immune recognition of nascent tumorigenic cells.

Weak immunogenicity of tumor cells is a root cause for the ultimate failure of immunosurveillance and immunotherapy. Although tumor evolution can be shaped by immunoediting toward a less immunogenic phenotype, mechanisms governing the initial immunogenicity of primordial tumor cells or original cancer stem cells remain obscure. Here, using a single tumor-repopulating cell (TRC) to form tumors in immunodeficient or immunocompetent mice, we demonstrated that immunogenic heterogeneity is an inherent trait of tumorigenic cells defined by the activation status of signal transducer and activator of transcription 1 (STAT1) protein in the absence of immune pressure. Subsequent investigation identified that the RNA binding protein cold shock domain-containing protein E1 (CSDE1) can promote STAT1 dephosphorylation by stabilizing T cell protein tyrosine phosphatase (TCPTP). A methyltransferase SET and MYN domain-containing 3 (SMYD3) was further identified to mediate H3K4 trimethylation of CSDE1 locus, which was under the regulation of mechanotransduction by cell-matrix and cell-cell contacts. Thus, owing to the differential epigenetic modification and subsequent differential expression of CSDE1, nascent tumorigenic cells may exhibit either a high or low immunogenicity. This identified SMYD3-CSDE1 pathway represents a potential prognostic marker for cancer immunotherapy effectiveness that requires further investigation.

The Role of Oxidative Inactivation of Phosphatase PTEN and TCPTP in Fatty Liver Disease.

Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are becoming increasingly prevalent worldwide. Despite the different etiologies, their spectra and histological feature are similar, from simple steatosis to more advanced stages such as steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Studies including peroxiredoxin knockout models revealed that oxidative stress is crucial in these diseases, which present as consequences of redox imbalance. Protein tyrosine phosphatases (PTPs) are a superfamily of enzymes that are major targets of reactive oxygen species (ROS) because of an oxidation-susceptible nucleophilic cysteine in their active site. Herein, we review the oxidative inactivation of two tumor suppressor PTPs, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and T-cell protein tyrosine phosphatase (TCPTP), and their contribution to the pathogenicity of ALD and NAFLD, respectively. This review might provide a better understanding of the pathogenic mechanisms of these diseases and help develop new therapeutic strategies to treat fatty liver disease.

Discovery of a selective TC-PTP degrader for cancer immunotherapy.

T-cell protein tyrosine phosphatase (TC-PTP), encoded by PTPN2, has emerged as a promising target for cancer immunotherapy. TC-PTP deletion in B16 melanoma cells promotes tumor cell antigen presentation, while loss of TC-PTP in T-cells enhances T-cell receptor (TCR) signaling and stimulates cell proliferation and activation. Therefore, there is keen interest in developing TC-PTP inhibitors as novel immunotherapeutic agents. Through rational design and systematic screening, we discovered the first highly potent and selective TC-PTP PROTAC degrader, TP1L, which induces degradation of TC-PTP in multiple cell lines with low nanomolar DC50s and >110-fold selectivity over the closely related PTP1B. TP1L elevates the phosphorylation level of TC-PTP substrates including pSTAT1 and pJAK1, while pJAK2, the substrate of PTP1B, is unaffected by the TC-PTP degrader. TP1L also intensifies interferon gamma (IFN-γ) signaling and increases MHC-I expression. In Jurkat cells, TP1L activates TCR signaling through increased phosphorylation of LCK. Furthermore, in a CAR-T cell and KB tumor cell co-culture model, TP1L enhances CAR-T cell mediated tumor killing efficacy through activation of the CAR-T cells. Thus, we surmise that TP1L not only provides a unique opportunity for in-depth interrogation of TC-PTP biology but also serves as an excellent starting point for the development of novel immunotherapeutic agents targeting TC-PTP.

Application of hybrid biophysical-biochemical methods to unravel the molecular basis for auto-inhibition and activation of protein tyrosine phosphatase TCPTP/PTPN2.

Since the discovery of protein tyrosine phosphorylation as one of the critical post-translational modifications, it has been well known that the activity of protein tyrosine kinases (PTKs) is tightly regulated. On the other hand, protein tyrosine phosphatases (PTPs) are often regarded to act constitutively active, but recently we and others have shown that many PTPs are expressed in an inactive form due to allosteric inhibition by their unique structural features. Furthermore, their cellular activity is highly regulated in a spatiotemporal manner. In general, PTPs share a conserved catalytic domain comprising about 280 residues that is flanked by either an N-terminal or a C-terminal non-catalytic segment, which differs significantly in size and structure from each other and is known to regulate specific PTP's catalytic activity. The well-characterized non-catalytic segments can be globular or intrinsically disordered. In this work, we have focused on the T-Cell Protein Tyrosine Phosphatase (TCPTP/PTPN2) and demonstrated how the hybrid biophysical-biochemical methods can be applied to unravel the underlying mechanism through which TCPTP's catalytic activity is regulated by the non-catalytic C-terminal segment. Our analysis showed that TCPTP is auto-inhibited by its intrinsically disordered tail and trans-activated by Integrin alpha-1's cytosolic region.

Selective Inhibition of PTP1B by New Anthraquinone Glycosides from Knoxia valerianoides.

Protein tyrosine phosphatase 1B (PTP1B) is highly validated as a therapeutic target for type 2 diabetes. However, active site-directed PTP1B inhibitors generally suffer from poor selectivity and bioavailability. Inspired by the identification of a unique anthraquinone-coumarin hybrid from Knoxia valerianoides exhibiting good specificity for PTP1B over the highly homologous T-cell protein tyrosine phosphatase (TCPTP), further chemical investigation of this plant species led to the isolation of nine new anthraquinone glycosides (1-9) and two known ones (10 and 11). Structures were characterized by a combination of spectroscopic analyses and chemical methods. All compounds showed PTP1B inhibitory activities with IC50 values ranging from 1.05 to 13.74 μM. Compounds 4 and 8 exhibited greater than 64-fold selectivity over TCPTP. Enzyme kinetic studies revealed that compounds 4 and 7 behaved as mixed-type inhibitors. Docking studies predicted similar binding modes of these compounds at the allosteric site positioned between helices α3 and α6.

Macromolecular crowding amplifies allosteric regulation of T-cell protein tyrosine phosphatase

T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of T-cell receptor and oncogenic receptor tyrosine kinase signaling and implicated in cancer and autoimmune disease. TC-PTP activity is modulated by an intrinsically disordered C-terminal region (IDR) and suppressed in cells under basal conditions. Invitro structural studies have shown that the dynamic reorganization of IDR around the catalytic domain, driven by electrostatic interactions, can lead to TC-PTP activity inhibition; however, the process has not been studied in cells. Here, by assessing a mutant (378KRKRPR383 mutated into 378EAAAPE383, called TC45E/A) with impaired tail-PTP domain interaction, we obtained evidence that the downmodulation of TC-PTP enzymatic activity by the IDR occurs in cells. However, we found that the regulation of TC-PTP by the IDR is only recapitulated invitro when crowding polymers that mimic the intracellular environment are present in kinetic assays using a physiological phosphopeptide. Our FRET-based assays invitro and in cells confirmed that the effect of the mutant correlates with an impairment of the intramolecular inhibitory remodeling of TC-PTP by the IDR. This work presents an early example of the allosteric regulation of a protein tyrosine phosphatase being controlled by the cellular environment and provides a framework for future studies and targeting of TC-PTP function.

PTPN2 in the Immunity and Tumor Immunotherapy: A Concise Review.

PTPN2 (protein tyrosine phosphatase non-receptor 2), also called TCPTP (T cell protein tyrosine phosphatase), is a member of the PTP family signaling proteins. Phosphotyrosine-based signaling of this non-transmembrane protein is essential for regulating cell growth, development, differentiation, survival, and migration. In particular, PTPN2 received researchers’ attention when Manguso et al. identified PTPN2 as a cancer immunotherapy target using in vivo CRISPR library screening. In this review, we attempt to summarize the important functions of PTPN2 in terms of its structural and functional properties, inflammatory reactions, immunomodulatory properties, and tumor immunity. PTPN2 exerts synergistic anti-inflammatory effects in various inflammatory cells and regulates the developmental differentiation of immune cells. The diversity of PTPN2 effects in different types of tumors makes it a potential target for tumor immunotherapy.