Protein Tyrosine Phosphatase Activity Research Articles

Development of Novel Phosphonodifluoromethyl-Containing Phosphotyrosine Mimetics and a First-In-Class, Potent, Selective, and Bioavailable Inhibitor of Human CDC14 Phosphatases.

Together with protein tyrosine kinases, protein tyrosine phosphatases (PTPs) control protein tyrosine phosphorylation and regulate numerous cellular functions. Dysregulated PTP activity is associated with the onset of multiple human diseases. Nevertheless, understanding of the physiological function and disease biology of most PTPs remains limited, largely due to the lack of PTP-specific chemical probes. In this study, starting from a well-known nonhydrolyzable phosphotyrosine (pTyr) mimetic, phosphonodifluoromethyl phenylalanine (F2Pmp), we synthesized 7 novel phosphonodifluoromethyl-containing bicyclic/tricyclic aryl derivatives with improved cell permeability and potency toward various PTPs. Furthermore, with fragment- and structure-based design strategies, we advanced compound 9 to compound 15, a first-in-class, potent, selective, and bioavailable inhibitor of human CDC14A and B phosphatases. This study demonstrates the applicability of the fragment-based design strategy in creating potent, selective, and bioavailable PTP inhibitors and provides a valuable probe for interrogating the biological roles of hCDC14 phosphatases and assessing their potential for therapeutic interventions.

Abstract 6972: Programmed death-ligand 1 (PD-L1) upregulates c-MET phosphorylation via protein tyrosine phosphatase and contributes to the development of MET amplification in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer

Abstract High expression of programmed death-ligand 1(PD-L1) is associated with poor survival outcomes in epidermal growth factor receptor (EGFR) mutation non-small cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors (EGFR-TKIs). In this study we aimed to determine how increased PD-L1 affects the signal transduction of EGFR-mutant NSCLC cells. We first established PD-L1 overexpression EGFR mutant NSCLC cells, and discovered that PD-L1 overexpression cells had upregulated c-MET phosphorylation compared with parental cells using receptor tyrosine kinase array study. Subsequently, we demonstrated that knocking out PD-L1 (CD 274) in EGFR mutant NSCLC cells resulted in down-regulation of c-MET phosphorylation. Since protein tyrosine phosphatases (PTP) are known key regulators for c-MET phosphorylation and dephosphorylation, the activity of PTP was assessed. In PD-L1 overexpression EGFR mutant NSCLC cells, the PTP activity was decreased, whereas in PD-L1 knock-out cells, the PTP activity was increased. Additionally, the downregulation of c-MET phosphorylation in PD-L1 knock-out cells could be restored by adding PTP inhibitor (Na3VO4). The gene expression analysis showed that PTP expression was down-regulated in PD-L1 overexpression cells, indicating that PD-L1 upregulates c-MET phosphorylation by reducing PTP expression. We further established xenograft animal models using NSCLC cells with strong baseline c-MET phosphorylation to determine the effect of c-MET phosphorylation on osimertinib (a 3rd-generation EGFR-TKI) treatment. We found that tumor cells with strong c-MET phosphorylation were prone to develop MET amplification as acquired resistance under osimertinib, which could be overcome with combination of osimertinib and tepotinib, a c-MET inhibitor. Taken together, our results expand our understanding of PD-L1’s role beyond an immune checkpoint in EGFR mutant NSCLC. PD-L1-regulated MET alterations could be a crucial mechanism leading to poor treatment outcome of EGFR-TKIs in PD-L1 overexpression EGFR mutant NSCLC. Citation Format: Derek De-Rui Huang, Chia-Chi Hsu, Wei-Hsun Hsu, Bin-Chi Liao, James Chih-Hsin Yang. Programmed death-ligand 1 (PD-L1) upregulates c-MET phosphorylation via protein tyrosine phosphatase and contributes to the development of MET amplification in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6972.

Preparation of Oxidized and Reduced PTP4A1 for Structural and Functional Studies.

The formation of a reversible disulfide bond between the catalytic cysteine and a spatially neighboring cysteine (backdoor) in protein tyrosine phosphatases (PTPs) serves as a critical regulatory mechanism for maintaining the activity of protein tyrosine phosphatases. The failure of such protection results in the formation of irreversibly oxidized cysteines into sulfonic acid in a highly oxidative cellular environment in the presence of free radicals. Hence, it is important to develop methods to interconvert PTPs into reduced and oxidized forms to understand their catalytic function in vitro. Protein tyrosine phosphatase 4A type 1 (PTP4A1), a dual-specificity phosphatase, is catalytically active in the reduced form. Unexpectedly, also its oxidized form performs a key biological function in systemic sclerosis (SSc) by forming a kinase-phosphatase complex with Src kinases. Thus, we developed simple and efficient protocols for producing oxidized and reduced PTP4A1 to elucidate their biological function, which can be extended to study other protein tyrosine phosphatases and other recombinantly produced proteins.

Alteration of circulating ACE2-network related miRNAs in patients with COVID-19

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Medical University of Warsaw and São Paulo Research Foundation (FAPESP). Introduction Coronavirus disease 2019 (COVID-19) is associated with an increased risk of mortality and adverse cardiovascular (CV) occurrences, especially in a group of patients with CV diseases (CVD). SARS-CoV-2 virus enters cells through angiotensin converting enzyme 2 (ACE2) cell membrane enzymes, leading to the downregulation in the ACE2-related pathways. Based on our previous in silico [1] results we hypothesized that alteration in the ACE2 interaction network caused by virus infection can affect the expression of miRNAs and influence severity of the disease. Purpose In this study we aimed to analyse the diagnostic and predictive utility of ACE2-related miRNAs, which were identified in our previous in silico analysis (miR-10b-5p, miR-124-3p, miR-200b-3p, miR-26b-5p, miR-302c-5p) in patients with COVID-19. We also aimed to unravel the functions of analyzed miRNAs, by using machine learning based tools, including SHAP for clinical data analysis, and bioinformatic tools for data mining, target predictions and enrichment analyses. Methods Blood samples and clinical data of 79 COVID-19 patients were collected at three different time points, including the day of admission, 7-days and 21-days after admission, as well as one time-point of 32 healthy volunteers. The composite endpoint was hospitalisation length of stay (>21 days) and/or death in follow-up. Results Delta low miR-200b-3p expression (after 7-days of admission) presents predictive utility in assessment of the hospital length of stay and/or death in ROC curve analysis (AUC:0.730, p=0.002) (Fig. 1). Delta low miR-200b-3p expression, together with diabetes mellitus (DM) are independent predictors of increased hospital length of stay and/or death (OR= 5.775; CI= 1.572- 21.214; p= 0.008, OR= 4.888; CI= 1.001- 23.858; p= 0.050, respectively). The expression levels of miR-26b-5p and miR-10b-5p in COVID-19 patients were found lower at the baseline, 7 and 21-days after admission compared to the healthy controls (p<0.0001 for all time points). SHAP analysis indicated levels of miR 200b-3p (day 7th), miR-302c-5p (day 7th), CRP (day 7th), neutrophils (day 0), and D-Dimer (day 0) as the most promising predictors of long hospitalisation for the COVID-19 patients. Pathway enrichment analysis showed that among top shared pathways between targets of analyzed miRNAs were IL-2 signaling pathway, and Pathways in cancer. miR-200b-3p showed regulation of COVID-19-related targets associated with T cell protein tyrosine phosphatase and HIF-1 transcriptional activity in hypoxia, key pathways in COVID-19. Bioinformatics analysis pointed out the role of those miRNAs in multiple CVD phenotypes associated with COVID-19 (Fig. 2). Conclusions In this study we validated and characterized miRNAs which could serve as novel, predictive biomarkers of the COVID-19 long term hospitalisation and can be used for early stratification of patients and prediction of severity of infection development in an individual.

TULA Proteins in Men, Mice, Hens, and Lice: Welcome to the Family.

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of TULA proteins in various metazoan taxa, for identifying potential roles of TULA-family proteins outside of their functions already established in mammalian systems, is examined.

The Sts Proteins: Modulators of Host Immunity.

The suppressor of TCR signaling (Sts) proteins, Sts-1 and Sts-2, are a pair of closely related signaling molecules that belong to the histidine phosphatase (HP) family of enzymes by virtue of an evolutionarily conserved C-terminal phosphatase domain. HPs derive their name from a conserved histidine that is important for catalytic activity and the current evidence indicates that the Sts HP domain plays a critical functional role. Sts-1HP has been shown to possess a readily measurable protein tyrosine phosphatase activity that regulates a number of important tyrosine-kinase-mediated signaling pathways. The in vitro catalytic activity of Sts-2HP is significantly lower than that of Sts-1HP, and its signaling role is less characterized. The highly conserved unique structure of the Sts proteins, in which additional domains, including one that exhibits a novel phosphodiesterase activity, are juxtaposed together with the phosphatase domain, suggesting that Sts-1 and -2 occupy a specialized intracellular signaling niche. To date, the analysis of Sts function has centered predominately around the role of Sts-1 and -2 in regulating host immunity and other responses associated with cells of hematopoietic origin. This includes their negative regulatory role in T cells, platelets, mast cells and other cell types, as well as their less defined roles in regulating host responses to microbial infection. Regarding the latter, the use of a mouse model lacking Sts expression has been used to demonstrate that Sts contributes non-redundantly to the regulation of host immunity toward a fungal pathogen (C. albicans) and a Gram-negative bacterial pathogen (F. tularensis). In particular, Sts-/- animals demonstrate significant resistance to lethal infections of both pathogens, a phenotype that is correlated with some heightened anti-microbial responses of phagocytes derived from mutant mice. Altogether, the past several years have seen steady progress in our understanding of Sts biology.

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.

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.

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.

Lead suppresses perlecan expression via EGFR-ERK1/2-COX-2-PGI2 pathway in cultured bovine vascular endothelial cells.

Vascular endothelial cell growth is essential for the repair of intimal injury. Perlecan, a large heparan sulfate proteoglycan, intensifies fibroblast growth factor-2 (FGF-2) signaling as a co-receptor for FGF-2 and its receptor, and promotes the proliferation of vascular endothelial cells. Previously, we reported that 2 µM of lead, a toxic heavy metal, downregulated perlecan core protein expression and then suppressed the growth of vascular endothelial cells. However, since the mechanisms involved in the repression of perlecan by lead remains unclear, we analyzed its detailed signaling pathway using cultured bovine aortic endothelial cells. Our findings indicate that 2 µM of lead inhibited protein tyrosine phosphatase (PTP) activity and induced cyclooxygenase-2 (COX-2) via phosphorylation of the epidermal growth factor receptor (EGFR) and its downstream extracellular signal-regulated kinases (ERK1/2). In addition, among the prostanoids regulated by COX-2, prostaglandin I2 (PGI2) specifically contributes to the downregulation of perlecan expression by lead. This study revealed an intracellular pathway-the EGFR-ERK1/2-COX-2-PGI2 pathway activated by inhibition of PTP by lead-as a pathway that downregulates endothelial perlecan synthesis. The pathway is suggested to serve as a mechanism for the repression of perlecan expression, which leads to a delay in cell proliferation by lead.

Expression of tyrosine phosphatases in relation to PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori

Protein tyrosine phosphorylation is a reversible, dynamic process regulated by the activities of tyrosine kinases and tyrosine phosphatases. Although the involvement of tyrosine kinases in the prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs) has been documented, few studies have been conducted on the involvement of protein tyrosine phosphatases (PTPs) in PTTH-stimulated ecdysteroidogenesis. In the present study, we investigated the correlation between PTPs and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that the basal PTP enzymatic activities exhibited development-specific changes during the last larval instar and pupation stage, with high activities being detected during the later stages of the last larval instar. PTP enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Pretreatment with phenylarsine oxide (PAO) and benzylphosphonic acid (BPA), two chemical inhibitors of tyrosine phosphatase, reduced PTTH-stimulated enzymatic activity. Determination of ecdysteroid secretion showed that treatment with PAO and BPA did not affect basal ecdysteroid secretion, but greatly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PTP activity is indeed involved in ecdysteroid secretion. PTTH-stimulated phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) was partially inhibited by pretreatment with either PAO or BPA, indicating the potential link between PTPs and phosphorylation of ERK and 4E-BP. In addition, we also found that in vitro treatment with 20-hydroxyecdysone did not affect PTP enzymatic activity. We further investigated the expressions of two important PTPs (PTP 1B (PTP1B) and the phosphatase and tension homologue (PTEN)) in Bombyx PGs. Our immunoblotting analysis showed that B. mori PGs contained the proteins of PTP1B and PTEN, with PTP1B protein undergoing development-specific changes. Protein levels of PTP1B and PTEN were not affected by PTTH treatment. The gene expression levels of PTP1B and PTEN showed development-specific changes. From these results, we suggest that PTTH-regulated PTP signaling may crosstalk with ERK and target of rapamycin (TOR) signaling pathways and is a necessary component for stimulation of ecdysteroid secretion.

Targeted OUM1/PTPRZ1 silencing and synergetic CDT/enhanced chemical therapy toward uveal melanoma based on a dual-modal imaging-guided manganese metal–organic framework nanoparticles

Metastasis and chemical resistance are the most serious problems in the treatment of highly aggressive uveal melanoma (UM). The newly identified lncRNA OUM1 is overexpressed in UM, functions as a catalyst and regulates protein tyrosine phosphatase (PTP) activity by binding to PTP receptor type Z1 (PTPRZ1), which plays an important role in cell proliferation, metastasis and chemotherapy resistance in the UM microenvironment. Hence, siRNAs that selectively knocking down the lncRNA OUM1 (siOUM1) and its target gene PTPRZ1 (siPTPRZ1) were designed to inhibit the OUM1/PTPRZ1 pathway to reduce PTP activity, and this reduction in activity interrupts protein tyrosine phosphorylation, suppresses UM proliferation and metastasis and improves cisplatin sensitivity in UM cells. Then, to overcome the limitations of the difficulty of drug administration and traditional therapeutics, the indocyanine green (ICG)-labeled manganese metal–organic framework (MOF) nanoparticles (NPs) were fabricated and linked with arginine-glycine-aspartate (RGD) peptide to carry siOUM1/siPTPRZ1 and cisplatin to achieve targeted siRNA interference-mediated therapy, enhanced cisplatin therapy and chemodynamic therapy. This NP system also has a dual-modal imaging ability because ICG is a near-infrared region fluorescent dye and manganese has the potential to be used in magnetic resonance imaging. This study verifies the significance of the newly discovered lncRNA OUM1 as a new therapeutic target for aggressive UM and provides a drug delivery NP system for precise treatment of UM accompanied with a dual-modal imaging ability.Graphical

Phosphorylation/dephosphorylation of PTP-PEST at Serine 39 is crucial for cell migration.

We investigated the molecular details of the role of protein tyrosine phosphatase (PTP)-PEST in cell migration. PTP-PEST knockout mouse embryonic fibroblasts (KO MEFs) and MEF cells expressing a dominant-negative mutant of PTP-PEST showed significant suppression of cell migration compared to MEF cells expressing wild-type PTP-PEST (WT MEFs). Moreover, MEF cells harbouring a constitutively active mutant of PTP-PEST (S39A MEFs) showed a marked decrease in cell migration. In addition, MEF cells with no PTP-PEST or little PTP activity rapidly adhered to fibronectin and made many focal adhesions compared to WT MEF cells. In contrast, S39A MEF cells showed weak adhesion to fibronectin and formed a few focal adhesions. Furthermore, investigating the subcellular localization showed that Ser39-phosphorylated PTP-PEST was favourably situated in the adherent area of the pseudopodia. Therefore, we propose that suppression of PTP-PEST enzyme activity due to Ser39-phosphorylation in pseudopodia and at the leading edge of migrating cells induces rapid and good adherence to the extracellular matrix. Thus, suppression of PTP activity by Ser39-phosphorylation is critical for cell migration. Three amino acid substitutions in human PTP-PEST have been previously reported to alter PTP activity. These amino acid substitutions in mouse PTP-PEST altered the migration of MEF cells in a positive correlation.

Activation of SH2 domain-containing protein tyrosine phosphatase and inflammatory expression in psoriasis

Psoriasis is a chronic autoimmune disease characterized by abnormal proliferation and differentiation of keratinocytes and infiltration of inflammatory cells into the site of inflammation. Plaque psoriasis is the most common type of psoriasis, affecting up to 80–90% of psoriasis cases. Among inflammatory cells, myeloid dendritic cells or Langerhans cells are mainly activated cells during the pathogenesis of psoriasis to induce activation and differentiation of naive T cells into T helper cells (Th)1 and Th17 cells. SH2 domain-containing protein tyrosine phosphatase (SHP) is a negative regulator of the phosphorylation of several proteins involved in cellular differentiation, growth and activation. Chronic inflammation promotes tumor progression, which is characterized by the release of carcinogenic antigens, including alpha-fetoprotein (AFP) and cancer antigen 125 (CA125) into blood and urine. They are common tumor markers to serve as predictors of cancer development and survival of cancer patients. To this end, blood samples of 103 psoriasis patients and 46 healthy subjects were collected. The mRNA expressions of SHP1 and SHP2 were examined by using quantitative RT-PCR and the serum levels of IL-6, TNF-α, IFN-γ, IL-17A, AFP and CA125 by ELISA. As a result, the mRNA level of SHP1 was higher expressed, whereas the level of SHP2 was unaltered in the patient group compared to the control individuals. Importantly, psoriasis patients had CA125 level higher than the clinical cutoff 35U/mL was 15.6%, while healthy individuals had CA125 level lower than 35U/mL. In addition, the serum TNF-α and IL-17A concentrations were significantly increased in the patient group. In conclusion, the results indicated the significant differences in expression of SHP1 gene and inflammatory response in psoriasis patients. This study further hint for investigations on the functional role of SHP1 in regulating activation of immune cells present in psoriasis patients.

Post-transcriptional regulatory feedback encodes JAK-STAT signal memory of interferon stimulation.

Immune cells fine tune their responses to infection and inflammatory cues. Here, using live-cell confocal microscopy and mathematical modelling, we investigate interferon-induced JAK-STAT signalling in innate immune macrophages. We demonstrate that transient exposure to IFN-γ stimulation induces a long-term desensitisation of STAT1 signalling and gene expression responses, revealing a dose- and time-dependent regulatory feedback that controls JAK-STAT responses upon re-exposure to stimulus. We show that IFN-α/β1 elicit different level of desensitisation from IFN-γ, where cells refractory to IFN-α/β1 are sensitive to IFN-γ, but not vice versa. We experimentally demonstrate that the underlying feedback mechanism involves regulation of STAT1 phosphorylation but is independent of new mRNA synthesis and cognate receptor expression. A new feedback model of the protein tyrosine phosphatase activity recapitulates experimental data and demonstrates JAK-STAT network’s ability to decode relative changes of dose, timing, and type of temporal interferon stimulation. These findings reveal that STAT desensitisation renders cells with signalling memory of type I and II interferon stimulation, which in the future may improve administration of interferon therapy.

Ultraviolet Radiation Exposure and its Impacts on Cutaneous Phosphorylation Signaling in Carcinogenesis: Focusing on Protein Tyrosine Phosphatases†.

Sunlight exposure is a significant risk factor for UV-induced deteriorating transformations of epidermal homeostasis leading to skin carcinogenesis. The ability of UVB radiation to cause melanoma, as well as basal and squamous cell carcinomas, makes UVB the most harmful among the three known UV ranges. UVB-induced DNA mutations and dysregulation of signaling pathways contribute to skin cancer formation. Among various signaling pathways modulated by UVB, tyrosine phosphorylation signaling which is mediated by the action of protein tyrosine kinases (PTKs) on specific tyrosine residues is highly implicated in photocarcinogenesis. Following UVB irradiation, PTKs get activated and their downstream signaling pathways contribute to photocarcinogenesis by promoting the survival of damaged keratinocytes and increasing cell proliferation. While UVB activates oncogenic signaling pathways, it can also activate tumor suppressive signaling pathways as initial protective mechanisms to maintain epidermal homeostasis. Tyrosine dephosphorylation is one of the protective mechanisms and is mediated by the action of protein tyrosine phosphatases (PTPs). PTP can counteract UVB-mediated PTK activation and downregulate oncogenic signaling pathways. However, PTPs have not been studied extensively in photocarcinogenesis with previous studies regarding their inactivation induced by UVB. This current review will summarize the recent progress in the protective function of PTPs in epidermal photocarcinogenesis.

Inhibitors of recombinant protein-tyrosine phosphatase 1B (PTP1B) from Khaya senegalensis: Towards a strategic target for therapeutic intervention in trypanosomiasis

Background: Tyrosine dephosphorylation, catalyzed by protein-tyrosine phosphatase (PTP), prevents trypanosome differentiation to a procyclic form that lacks diverse mechanisms for survival within the mammalian host's bloodstream. Thus, differentiation to a procyclic form in the mammalian host's bloodstream would be potentially lethal to the parasite. This demonstrates that PTP is a critical regulator of trypanosome differentiation, making it a strategic therapeutic target for trypanosomiasis.Purpose: The in vitro inhibitory effect of seven compounds from the active fraction of Khaya senegalensis stem bark (4‑hydroxy-2-butanone, 1,3-butanediol, 2-methylpropyl butanoate, n-tridecanoic acid (CH3-(CH2)11-COOH), n-tetratriacontane, n-hexadecanoic (CH3-(CH2)14-COOH), and 14-pentadecenoic acid (CH2=CH-(CH2)12-COOH)) on the enzymatic activity of recombinant human PTP1B (MBL-SE332–0050) was investigated in this study.Method: The inhibition study was performed according to the standard procedure for analytical experiment and was monitored spectrophotometrically. PTP1B was assayed with 10 mM of p-nitrophenylphosphate. Bioassay-guided fractionation of methanol stem bark extract from K. senegalensis yielded the potent PTP1B inhibitors. Sodium orthovanadate was used as the positive control for inhibition. The kinetic parameters were determined by Lineweaver-Burk plots and calculated using Sigma Plot (SPCC Inc., Chicago, IL, USA)Results: The active fraction that contains the seven compounds inhibited PTP1B dose-dependently with an IC50 of 1.32 µM. Lineweaver-Burk plots revealed that PTP1B was inhibited non-competitively with a Ki of 0.46 ± 0.03 mg/ml, while sodium orthovanadate inhibited the enzyme competitively with an IC50 of 1.02 µM and Ki of 0.26 ± 0.01 mg/ml. Cleavage of the substrate para-Nitrophenylphosphate (pNPP) by PTP1B showed a KM of 4.99 mM and Vmax of 0.053 µmol/min.Conclusion: The mixture of compounds tested is potent PTP1B inhibitors that could serve as scaffolds for new trypanocidal drug candidates that target protein-tyrosine phosphatase activity.

Biochemical, Enzymatic, and Computational Characterization of Recurrent Somatic Mutations of the Human Protein Tyrosine Phosphatase PTP1B in Primary Mediastinal B Cell Lymphoma.

Human protein tyrosine phosphatase 1B (PTP1B) is a ubiquitous non-receptor tyrosine phosphatase that serves as a major negative regulator of tyrosine phosphorylation cascades of metabolic and oncogenic importance such as the insulin, epidermal growth factor receptor (EGFR), and JAK/STAT pathways. Increasing evidence point to a key role of PTP1B-dependent signaling in cancer. Interestingly, genetic defects in PTP1B have been found in different human malignancies. Notably, recurrent somatic mutations and splice variants of PTP1B were identified in human B cell and Hodgkin lymphomas. In this work, we analyzed the molecular and functional levels of three PTP1B mutations identified in primary mediastinal B cell lymphoma (PMBCL) patients and located in the WPD-loop (V184D), P-loop (R221G), and Q-loop (G259V). Using biochemical, enzymatic, and molecular dynamics approaches, we show that these mutations lead to PTP1B mutants with extremely low intrinsic tyrosine phosphatase activity that display alterations in overall protein stability and in the flexibility of the active site loops of the enzyme. This is in agreement with the key role of the active site loop regions, which are preorganized to interact with the substrate and to enable catalysis. Our study provides molecular and enzymatic evidence for the loss of protein tyrosine phosphatase activity of PTP1B active-site loop mutants identified in human lymphoma.

Preferential redox regulation of cysteine-based protein tyrosine phosphatases: structural and biochemical diversity.

Protein phosphorylation is a major post-translational modification involved in cell signalling that regulates many physiological and pathological processes. Despite their biological importance, protein phosphatases are less studied than protein kinases. Importantly, the activity of Cys-based protein tyrosine phosphatases (PTPs) can be regulated by reversible oxidation. The initial two-electron oxidation product of the active site Cys is a sulfenic acid (Cys-SOH) that can then undergo distinct outcomes, such as the disulfide bond or a sulfenyl amide formation. Here, we review the biochemical and structural features of PTPs to find patterns that might specify their oxidation products, aiming to get insights into redox regulatory mechanisms. Initially, the structure and biochemistry of PTP1B is presented. Then, we describe structural aspects that are relevant for substrate recognition and catalysis. Notably, all PTPs contain critical Cys residues for the catalysis of dephosphorylation that is prone to oxidative inactivation, which are frequently found oxidized in cells under physiological conditions, such as upon growth factor stimuli. However, direct oxidations of Cys residues in PTPs by H2 O2 are rather slow. Therefore, we discuss possible mechanisms that may account for this apparent contradiction between biological and chemical redox aspects of PTPs. Furthermore, we performed a systematic analysis of the distance between active site cysteine and its backdoor cysteine with the attempt to analyse the preference between disulfide bond formation or sulfenyl amide interaction upon oxidation. In summary, PTPs have been showing many possibilities to auto-protect from irreversible oxidation, which is important for cell signalling regulation.

High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells.

Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, we report a microwell array format live-cell analysis device (LCAD) that can perform localized-electroporation induced membrane permeabilization, for cellular delivery or sampling, and directly interfaces with surface-based biosensors for analyzing the extracted contents. We demonstrate the capabilities of the LCAD via an automated high-throughput workflow for multimodal analysis of live-cell dynamics, consisting of quantitative measurements of enzyme activity using self-assembled monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced imaging and analysis. By combining a fabrication protocol that enables robust assembly and operation of multilayer devices with embedded gold electrodes and an automated imaging workflow, we successfully deliver functional molecules (plasmid and siRNA) into live cells at multiple time-points and track their effect on gene expression and cell morphology temporally. Furthermore, we report sampling performance enhancements, achieving saturation levels of protein tyrosine phosphatase activity measured from as few as 60 cells, and demonstrate control over the amount of sampled contents by optimization of electroporation parameters using a lumped model. Lastly, we investigate the implications of cell morphology on electroporation-induced sampling of fluorescent molecules using a deep-learning enhanced image analysis workflow.