In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase PTP-PEST.

The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPKα subunit. However, the identity of the phosphotyrosine residues that PTP-PEST dephosphorylates remains unknown. In this study, we first predicted the structure of the complex of the AMPKα2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirmed the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicated that residue Tyr232 is the most likely dephosphorylation site on AMPKα2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. Phosphorylation of the highly conserved Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. We generated a phosphomimetic (PTP-PEST-Y64D) mutant and used co-immunoprecipitation to study the effect of PTP-PEST phosphorylation on AMPKα2 binding. The mutant exhibited an increased affinity for AMPKα2 and corroborated the in-silico predictions. Together, our findings present a plausible structural basis of AMPK regulation by PTP-PEST and show how phosphorylation of PTP-PEST affects its interaction with AMPKα2.

Protein tyrosine phosphatase non-receptor type 12 suppresses tumor progression in osteosarcoma cells.

BACKGROUND: Protein tyrosine phosphatase non-receptor 12 (PTPN12) plays a prominent role in various cancers as a tumor suppressor. However, the expression of PTPN12 and its biological functions in osteosarcoma (OS) remains to be determined. METHODS: PTPN12 expression in OS was explored in public databases and detected by immunohistochemistry and Western blot. The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay and colony formation. The cell migration and invasion were assessed by the Transwell assay. Flow cytometry analysis was applied to detect cell apoptosis and cell cycle distribution. To investigate the related mechanism, the levels of EGFR and downstream proteins were detected by Western blot. RESULTS: PTPN12 expression was significantly decreased in OS samples in GEO database and our hospital. OS cell lines in Cancer Cell Line Encyclopedia (CCLE) database and our cultured OS cells also demonstrated low PTPN12 expression. Lentivirus-induced overexpression of PTPN12 significantly inhibited the cell viability, migration and invasion of 143B and U2OS cells. The results of flow cytometry found that PTPN12 overexpression promoted cell apoptosis and induced cell cycle arrest at G1 phase in 143B and U2OS cells. The phosphorylation levels of EGFR and subsequent proteins of the PI3K/AKT and ERK pathways were inactivated as a result of PTPN12 overexpression in OS. CONCLUSION: PTPN12 plays a tumor suppressive role in OS cells. Restoring of PTPN12 activity may provide new insights for the treatment of this disease.

Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates.

Colonization of host phagocytic cells by Leishmania metacyclic promastigotes involves several parasite effectors, including the zinc-dependent metalloprotease GP63. The major mode of action of this virulence factor entails the cleavage/degradation of host cell proteins. Given the potent proteolytic activity of GP63, identification of its substrates requires the adequate preparation of cell lysates to prevent artefactual degradation during cell processing. In the present study, we re-examined the cleavage/degradation of reported GP63 substrates when GP63 activity was efficiently neutralized during the preparation of cell lysates. To this end, we infected bone marrow-derived macrophages with either wild type, Δgp63, and Δgp63+GP63 L. major metacyclic promastigotes for various time points. We prepared cell lysates in the absence or presence of the zinc-metalloprotease inhibitor 1,10-phenanthroline and examined the levels and integrity of ten previously reported host cell GP63 substrates. Inhibition of GP63 activity with 1,10-phenanthroline during the processing of macrophages prevented the cleavage/degradation of several previously described GP63 targets, including PTP-PEST, mTOR, p65RelA, c-Jun, VAMP3, and NLRP3. Conversely, we confirmed that SHP-1, Synaptotagmin XI, VAMP8, and Syntaxin-5 are bona fide GP63 substrates. These results point to the importance of efficiently inhibiting GP63 activity during the preparation of Leishmania-infected host cell lysates. In addition, our results indicate that the role of GP63 in Leishmania pathogenesis must be re-evaluated.

Depletion of circ_0128846 ameliorates interleukin-1ß-induced human chondrocyte apoptosis and inflammation through the miR-940/PTPN12 pathway.

BACKGROUND: Previous evidence has suggested that circular RNA (circRNA) is abnormally expressed in osteoarthritis (OA). However, the underlying mechanism of circRNA in OA progression remains unclear. The study aims to reveal the mechanism of circ_0128846 regulating OA. METHODS: Human chondrocytes (C28/I2 cells) were treated with interleukin-1ß (IL-1ß) to mimic an OA cell model. The expression levels of circ_0128846, miR-940 and protein tyrosine phosphatase 12 (PTPN12) were detected by qRT-PCR. Protein expression was checked by Western blotting. Cell viability, proliferation, and apoptosis were analyzed by a cell counting kit-8 assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α) and IL-6 was determined by an enzyme-linked immunosorbent assay (ELISA). The binding relationship between miR-940 and circ_0128846 or PTPN12 was identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: Circ_0128846 and PTPN12 expression were significantly upregulated, whereas miR-940 was downregulated in the cartilage tissues of OA patients and IL-1ß-treated C28/I2 cells compared with controls. IL-1ß treatment inhibited C28/I2 cell proliferation and induced cell apoptosis and the production of inflammatory factors, TNF-α and IL-6; however, these effects were partly reversed after circ_0128846 depletion. In terms of mechanism, circ_0128846 acted as a miR-940 sponge, and miR-940 combined with PTPN12. Also, circ_0128846 depletion partly ameliorated IL-1ß-induced C28/I2 cell disorders through miR-940. PTPN12 overexpression also partly relieved miR-940-mediated effects in IL-1ß-treated C28/I2 cells. Further, circ_0128846 induced PTPN12 expression by interacting with miR-940. CONCLUSION: Circ_0128846 regulated human chondrocyte proliferation, apoptosis and inflammation through the miR-940/PTPN12 pathway in OA.

LncRNA GATA2-AS1 suppresses esophageal squamous cell carcinoma progression via the mir-940/PTPN12 axis.

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor worldwide. Long noncoding RNAs (lncRNAs) exhibit a regulatory role in the progression of ESCC. Our research was performed to investigate the potential molecular mechanism of lncRNA GATA2-AS1 in ESCC. METHODS: The expression of GATA2-AS1 was identified by qRT-PCR. Cell function assays explored the potential effect of GATA2-AS1 on ESCC progression. The subcellular hierarchical localization method was executed to identify the subcellular localization of GATA2-AS1 in ESCC cells. A prediction website was utilized to discover the relationships among GATA2-AS1, miR-940 and PTPN12. Dual luciferase reporter gene, pull-down assays and RIP assays were executed to verify the binding activity among GATA2-AS1, miR-940 and PTPN12. Xenograft tumor experiments were performed to evaluate ESCC cell growth in vivo. RESULTS: The expression of GATA2-AS1 and PTPN12 was reduced, while miR-940 expression was enhanced in ESCC tissues and cell lines. In vivo experiments showed that GATA2-AS1 inhibited the progression of ESCC cells toward malignancy. Bioinformatics analysis, dual luciferase and RIP assays revealed that GATA2-AS1 upregulated PTPN12 expression by competitively targeting miR-940. miR-940 reversed the inhibitory effect of GATA2-AS1 on the biological behavior of ESCC cells. CONCLUSION: Our findings suggested that GATA2-AS1, expressed at low levels in ESCC, plays a crucial role in the progression of ESCC by targeting the miR-940/PTPN12 axis and could be a potential drug target to treat ESCC patients.

SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding.

The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, e.g., Lck, Fyn, Lyn, Hck, Fgr, Blk, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that mediates autoinhibition. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (LYP/Pep), which dephosphorylate the SFK activation loop to promote autoinhibition. Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains, and high local concentration promotes Csk function. Purified Csk homodimerizes in solution through an interface that overlaps the phosphatase binding footprint. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Disruption of either interaction in cells, however, decreased the negative-regulatory function of Csk. Csk W47A, a substitution previously reported to block PTPN22 binding, had a secondary effect of impairing homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

Downregulation of secreted frizzled-related protein 4 inhibits hypoxia/reoxygenation injury in diabetic cardiomyocytes by protein tyrosine phosphatase nonreceptor type 12.

Myocardial ischemia-reperfusion injury in diabetic patients leads to an increased incidence of complications and mortality. Secreted frizzled-related protein 4 (SFRP4) plays a critical role in diabetic myocardial ischemia-reperfusion. This paper aims to uncover the underlying mechanisms of SFRP4 in hypoxia/reoxygenation (H/R) injury of diabetic myocardial cells. An in vitro ischemia/reperfusion (I/R) injury model was established using high glucose-induced H9c2 cardiomyocytes. Expression of SFRP4 was detected by real-time reverse transcriptase-polymerase chain reaction and Western blotting. After transfection of SFRP4, the binding of SFRP4 to protein tyrosine phosphatase nonreceptor type 12 (PTPN12) was predicted by database and verified by co-immunoprecipitation assay. P13 K/AKT protein levels were examined by Western blotting. PTPN12 levels were tested by RT-qPCR and Western blotting, cell viability by Cell Counting Kit-8, lactose dehydrogenase kit, terminal dUTP nick-end labeling assay, and cell inflammation and oxidative stress by Western blotting and enzyme linked immunosorbent assay. After overexpression of PTPN12, the experiments for cell viability, inflammation and oxidative stress were repeated once more. SFRP4 expression was upregulated in a high-glucose-stimulated H/R cardiomyocyte model. The interference of SFRP4 promoted cell viability, inhibited the inflammatory and oxidative stress response of H/R cardiomyocytes induced by high glucose. SFRP4 interacted with PTPN12 and inhibited the PI3K/AKT signaling pathway. PTPN12 overexpression reversed the inhibitory effect of sh-SFRP4 on H/R cardiomyocyte damage induced by high glucose. Downregulation of SFRP4 inhibited H/R cell damage in diabetic cardiomyocytes by binding to PTPN12.

Syntenin-1-mediated small extracellular vesicles promotes cell growth, migration, and angiogenesis by increasing onco-miRNAs secretion in lung cancer cells.

Small extracellular vesicles (sEVs) play a pivotal role in tumor progression by mediating intercellular communication in the tumor microenvironment (TME). Syntenin-1 induces malignant tumor progression in various types of human cancers, including human lung cancer and regulates biogenesis of sEVs. However, the function of syntenin-1-regulated sEVs and miRNAs in sEVs remains to be elucidated. In the present study, we aimed to demonstrate the role of oncogenic Ras/syntenin-1 axis in the release of sEVs and elucidate the function of syntenin-1-mediated miRNAs in sEVs in lung cancer progression. The results revealed that oncogenic Ras promoted the release of sEVs by inducing syntenin-1 expression; disruption of syntenin-1 expression impaired the release of sEVs as well as sEV-mediated cancer cell migration and angiogenesis. Moreover, we identified three miRNAs, namely miR-181a, miR-425-5p, and miR-494-3p, as onco-miRNAs loaded into syntenin-1-dependent sEVs. Remarkably, miR-494-3p was highly abundant in sEVs and its release was triggered by syntenin-1 expression and oncogenic Ras. Ectopic expression of the miR-494-3p mimic enhanced the migration and proliferation of lung cancer cells as well as tube formation in endothelial cells; however, the miR-494-3p inhibitor blocked sEV-mediated effects by targeting tyrosine-protein phosphatase nonreceptor type 12 (PTPN12), a tumor suppressor. sEVs promoted tumor growth and angiogenesis by downregulating PTPN12 expression; however, the miR-494-3p inhibitor significantly suppressed these effects in vivo, confirming that miR-494-3p acts as a major onco-miRNA loaded into lung cancer cell-derived sEVs. Eventually, the oncogenic Ras/syntenin-1 axis may induce cancer progression by increasing miR-494-3p loading into sEVs in lung cancer cells in the TME.

PTP-PEST Regulated Membranous/Cytoplasmic Translocation of p120ctn in the Lung Cancer Resistance to Tyrosine Kinase Inhibitor.

Our previous studies indicate that resistance induction using first-generation tyrosine kinase inhibitors (TKIs) in lung cancer is accompanied with p120-catenin (p120ctn) cytoplasmic translocation from the membrane. However, the molecular mechanism underlying p120ctn intracytoplasmic translocation has not yet been reported. We performed immunohistochemistry to detect the correlation of p120ctn distribution with protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) and p120ctn Y335 phosphorylation levels in non-small cell lung cancer (NSCLC) patients. After resistance induction using first-generation TKIs in lung cancer cells, Western blotting and substrate trapping were used to assess PTP-PEST expression and its influence on p120ctn Y335 phosphorylation, as well as the role of p120ctn Y335 phosphorylation on the association of p120ctn with E-cadherin and p120ctn membrane/cytoplasm translocation. In 197 samples collected from NSCLC patients, cytoplasmic p120ctn and enhanced p120ctn Y335 phosphorylation were associated with decreased PTP-PEST. After resistance induction using gefitinib, decreased PTP-PEST expression was accompanied by enhanced phosphorylation of p120ctn Y335 and p120ctn translocated to the cytoplasm. In gefitinib-resistant cells, PTP-PEST overexpression restrained p120ctn Y335 phosphorylation and restored membrane p120ctn expression. PTP-PEST enhanced the interaction of p120ctn with E-cadherin and elevated p120ctn membrane expression. However, increased p120ctn-Y335F mutant had no effect on p120ctn interaction with E-cadherin and membrane/cytoplasm translocation compared with the control group. In conclusion, resistance to first-generation TKIs inhibited PTP-PEST expression, which promoted p120ctn-Y335 phosphorylation and reduced the interaction of p120ctn with E-cadherin, resulting in p120ctn cytoplasmic translocation.

Protein tyrosine phosphatase non-receptor type 12 (PTPN12), negatively regulated by miR-106a-5p, suppresses the progression of hepatocellular carcinoma.

Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is abnormally expressed in many human cancers. However, its role in hepatocellular carcinoma (HCC) is indeterminate. In this study, immunohistochemistry and Western blot were adopted to detect PTPN12 protein expression in HCC tissues and cell lines. MiR-106a-5p and PTPN12 mRNA expressions were determined by quantitative real-time polymerase chain reaction (qRT-PCR). siRNA was used to knockdown PTPN12 expression in HCC cells, and the multiplication, migration, and invasion of HCC cells were determined by cell counting kit 8 (CCK-8) and Transwell assays. The interaction between PTPN12 and miR-106a-5p was verified by dual-luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. In the present study, we demonstrated that PTPN12 expression in HCC tissues and cells was significantly decreased, which was associated with the tumor size, TNM stage, and lymph node metastasis of HCC patients. Functionally, knocking down PTPN12 significantly promoted the multiplication, migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells. PTPN12 was identified as the direct target of miR-106a-5p, and its expression was negatively modulated by miR-106a-5p. Besides, PTPN12 counteracted the promoting effects of miR-106a-5p on the viability, migration, invasion, and EMT of HCC cells. In conclusion, this study substantiates that PTPN12 inhibits the growth, migration, invasion, and EMT of HCC cells, and miR-106a-5p contributes to its dysregulation in HCC.

circPTPN12/miR-21-5 p/∆Np63α pathway contributes to human endometrial fibrosis.

Emerging evidence demonstrates the important role of circular RNAs (circRNAs) in regulating pathological processes in various diseases including organ fibrosis. Endometrium fibrosis is the leading cause of uterine infertility, but the role of circRNAs in its pathogenesis is largely unknown. Here, we provide the evidence that upregulation of circPTPN12 in endometrial epithelial cells (EECs) of fibrotic endometrium functions as endogenous sponge of miR-21-5 p to inhibit miR-21-5 p expression and activity, which in turn results in upregulation of ΔNp63α to induce the epithelial mesenchymal transition (EMT) of EECs (EEC-EMT). In a mouse model of endometrium fibrosis, circPTPN12 appears to be a cofactor of driving EEC-EMT and administration of miR-21-5 p could reverse this process and improve endometrial fibrosis. Our findings revealed that the dysfunction of circPTPN12/miR-21-5 p/∆Np63α pathway contributed to the pathogenesis of endometrial fibrosis.

Spatially interacting phosphorylation sites and mutations in cancer.

Advances in mass-spectrometry have generated increasingly large-scale proteomics datasets containing tens of thousands of phosphorylation sites (phosphosites) that require prioritization. We develop a bioinformatics tool called HotPho and systematically discover 3D co-clustering of phosphosites and cancer mutations on protein structures. HotPho identifies 474 such hybrid clusters containing 1255 co-clustering phosphosites, including RET p.S904/Y928, the conserved HRAS/KRAS p.Y96, and IDH1 p.Y139/IDH2 p.Y179 that are adjacent to recurrent mutations on protein structures not found by linear proximity approaches. Hybrid clusters, enriched in histone and kinase domains, frequently include expression-associated mutations experimentally shown as activating and conferring genetic dependency. Approximately 300 co-clustering phosphosites are verified in patient samples of 5 cancer types or previously implicated in cancer, including CTNNB1 p.S29/Y30, EGFR p.S720, MAPK1 p.S142, and PTPN12 p.S275. In summary, systematic 3D clustering analysis highlights nearly 3,000 likely functional mutations and over 1000 cancer phosphosites for downstream investigation and evaluation of potential clinical relevance.

Progress in the correlation between PTPN12 gene expression and human tumors.

BACKGROUND: The global morbidity of cancer is rising rapidly. Despite advances in molecular biology, immunology, and cytotoxic and immune-anticancer therapies, cancer remains a major cause of death worldwide. Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a new member of the cytoplasmic protein tyrosine phosphatase family, isolated from a cDNA library of adult colon tissue. Thus far, no studies have reviewed the correlation between PTPN12 gene expression and human tumors. METHODS: This article summarizes the latest domestic and international research developments on how the expression of PTPN12 relates to human tumors. The extensive search in Web of Science and PubMed with the keywords including PTPN12, tumor, renal cell carcinoma, proto-oncogenes, tumor suppressor genes was undertaken. RESULTS: More and more studies have shown that a tumor is essentially a genetic disease, arising from a broken antagonistic function between proto-oncogenes and tumor suppressor genes. When their antagonistic effect is out of balance, it may cause uncontrolled growth of cells and lead to the occurrence of tumors. PTPN12 is a tumor suppressor gene, so inhibiting its activity will lead directly or indirectly to the occurrence of tumors. CONCLUSION: The etiology, prevention, and treatment of tumors have become the focus of research around the world. PTPN12 is a tumor suppressor gene. In the future, PTPN12 might serve as a novel molecular marker to benefit patients, and even the development of tumor suppressor gene activation agents can form a practical research direction.

Targeting protein tyrosine phosphatase PTP-PEST (PTPN12) for therapeutic intervention in acute myocardial infarction.

AIMS: The myocardial ischaemia/reperfusion (I/R) injury is almost inevitable since reperfusion is the only established treatment for acute myocardial infarction (AMI). To date there is no effective strategy available for reducing the I/R injury. Our aim was to elucidate the mechanisms underlying myocardial I/R injury and to develop a new strategy for attenuating the damage it causes. METHODS AND RESULTS: Using a mouse model established by ligation of left anterior descending artery, we found an increase in activity of protein tyrosine phosphatases (PTPs) in myocardium during I/R. Treating the I/R-mice with a pan-PTP inhibitor phenyl vinyl sulfone attenuated I/R damage, suggesting PTP activation to be harmful in I/R. Through analysing RNAseq data, we showed PTPs being abundantly expressed in mouse myocardium. By exposing primary cardiomyocytes ablated with specific endogenous PTPs by RNAi to hypoxia/reoxygenation (H/R), we found a role that PTP-PEST (PTPN12) plays to promote cell death under H/R stress. Auranofin, a drug being used in clinical practice for treating rheumatoid arthritis, may target PTP-PEST thus suppressing its activity. We elucidated the molecular basis for Auranofin-induced inactivation of PTP-PEST by structural studies, and then examined its effect on myocardial I/R injury. In the mice receiving Auranofin before reperfusion, myocardial PTP activity was suppressed, leading to restored phosphorylation of PTP-PEST substrates, including ErbB-2 that maintains the survival signalling of the heart. In line with the inhibition of PTP-PEST activity, the Auranofin-treated I/R-mice had smaller infarct size and better cardiac function. CONCLUSIONS: PTP-PEST contributes to part of the damages resulting from myocardial I/R. The drug Auranofin, potentially acting through the PTP-PEST-ErbB-2 signalling axis, reduces myocardial I/R injury. Based on this finding, Auranofin could be used in the development of new treatments that manage I/R injury in patients with AMI.

High-level expression of protein tyrosine phosphatase non-receptor 12 is a strong and independent predictor of poor prognosis in prostate cancer.

BACKGROUND: Protein tyrosine phosphatase non-receptor 12 (PTPN12) is ubiquitously tyrosine phosphatase with tumor suppressive properties. METHODS: PTPN12 expression was analyzed by immunohistochemistry on a tissue microarray with 13,660 clinical prostate cancer specimens. RESULTS: PTPN12 staining was typically absent or weak in normal prostatic epithelium but seen in the majority of cancers, where staining was considered weak in 26.5%, moderate in 39.9%, and strong in 4.7%. High PTPN12 staining was associated with high pT category, high classical and quantitative Gleason grade, lymph node metastasis, positive surgical margin, high Ki67 labeling index and early prostate specific antigen recurrence (p < 0.0001 each). PTPN12 staining was seen in 86.4% of TMPRSS2:ERG fusion positive but in only 58.4% of ERG negative cancers. Subset analyses discovered that all associations with unfavorable phenotype and prognosis were markedly stronger in ERG positive than in ERG negative cancers but still retained in the latter group. Multivariate analyses revealed an independent prognostic impact of high PTPN12 expression in all cancers and in the ERG negative subgroup and to a lesser extent also in ERG positive cancers. Comparison with 12 previously analyzed chromosomal deletions revealed that high PTPN12 expression was significantly associated with 10 of 12 deletions in ERG negative and with 7 of 12 deletions in ERG positive cancers (p < 0.05 each) indicating that PTPN12 overexpression parallels increased genomic instability in prostate cancer. CONCLUSIONS: These data identify PTPN12 as an independent prognostic marker in prostate cancer. PTPN12 analysis, either alone or in combination with other biomarkers might be of clinical utility in assessing prostate cancer aggressiveness.

Critical Structural Defects Explain Filamin A Mutations Causing Mitral Valve Dysplasia.

Mitral valve diseases affect ∼3% of the population and are the most common reasons for valvular surgery because no drug-based treatments exist. Inheritable genetic mutations have now been established as the cause of mitral valve insufficiency, and four different missense mutations in the filamin A gene (FLNA) have been found in patients suffering from nonsyndromic mitral valve dysplasia (MVD). The filamin A (FLNA) protein is expressed, in particular, in endocardial endothelia during fetal valve morphogenesis and is key in cardiac development. The FLNA-MVD-causing mutations are clustered in the N-terminal region of FLNA. How the mutations in FLNA modify its structure and function has mostly remained elusive. In this study, using NMR spectroscopy and interaction assays, we investigated FLNA-MVD-causing V711D and H743P mutations. Our results clearly indicated that both mutations almost completely destroyed the folding of the FLNA5 domain, where the mutation is located, and also affect the folding of the neighboring FLNA4 domain. The structure of the neighboring FLNA6 domain was not affected by the mutations. These mutations also completely abolish FLNA's interactions with protein tyrosine phosphatase nonreceptor type 12, which has been suggested to contribute to the pathogenesis of FLNA-MVD. Taken together, our results provide an essential structural and molecular framework for understanding the molecular bases of FLNA-MVD, which is crucial for the development of new therapies to replace surgery.

Clinical implications of a novel prognostic factor AIFM3 in breast cancer patients.

BACKGROUND: In a time of increasing concerns over personalized and precision treatment in breast cancer (BC), filtering prognostic factors attracts more attention. Apoptosis-Inducing Factor Mitochondrion-associated 3 (AIFM3) is widely expressed in various tissues and aberrantly expressed in several cancers. However, clinical implication of AIFM3 has not been reported in BC. The aim of the study is to investigate the crystal structure, clinical and prognostic implications of AIFM3 in BC. METHODS: AIFM3 expression in 151 BC samples were assessed by immunohistochemistry (IHC). The Cancer Genome Atlas (TCGA) and Kaplan-Meier survival analysis were used to demonstrate expression and survival of AIFM3 signature. Gene Set Enrichment Analysis (GSEA) was performed to investigate the mechanisms related to AIFM3 expression in BC. RESULTS: AIFM3 was significantly more expressed in breast cancer tissues than in normal tissues. AIFM3 expression had a significant association with tumor size, lymph node metastasis, TNM stage and molecular typing. Higher AIFM3 expression was related to a shorter overall survival (OS) and disease-free survival (DFS). Lymph node metastasis and TNM stage were independent factors of AIFM3 expression. The study presented the crystal structure of AIFM3 successfully and predicted several binding sites when AIFM3 bonded to PTPN12 by Molecular Operating Environment software (MOE). CONCLUSIONS: AIFM3 might be a potential biomarker for predicting prognosis in BC, adding to growing evidence that AIFM3 might interact with PTPN12.

A missense variant in PTPN12 associated with the risk of colorectal cancer by modifying Ras/MEK/ERK signaling.

BACKGROUND: The classical protein tyrosine phosphatases (PTPs) have been widely reported to be associated with various human malignancies including colorectal cancer (CRC). However, there are few comprehensive analyses of the association between the classical PTP genes and CRC risk. METHODS: First, a bioinformatics analysis was performed to identify missense variants within the classical PTP gene family. Second, exome-wide association data and an independent population study were conducted to evaluate effects of candidate variants on CRC risk. Finally, functional assays based on signaling pathways were applied to uncover the potential pathogenic mechanism. RESULTS: We identified that PTPN12 rs3750050 G allele presented a 19% increase the risk of CRC, with an OR of 1.19 (95% CI = 1.09-1.30, P = 1.015×10-4) under an additive model in the combined analysis. Furthermore, biochemical assays illustrated that rs3750050 could impair the inhibitory effect of PTPN12 on Ras/MEK/ERK signaling by impeding SHC dephosphorylation, increase the expression of cyclin D1 and ultimately lead to aberrant cell proliferation, thus contributing to CRC pathogenesis. CONCLUSION: Our study highlights that PTPN12 rs3750050 could increase CRC risk by modifying Ras/MEK/ERK signaling. This work provides a novel insight into the roles of genetic variants within PTP genes in the pathogenesis of CRC.

Non-syndromic Mitral Valve Dysplasia Mutation Changes the Force Resilience and Interaction of Human Filamin A.

Filamin A (FLNa), expressed in endocardial endothelia during fetal valve morphogenesis, is key in cardiac development. Missense mutations in FLNa cause non-syndromic mitral valve dysplasia (FLNA-MVD). Here, we aimed to reveal the currently unknown underlying molecular mechanism behind FLNA-MVD caused by the FLNa P637Q mutation. The solved crystal structure of the FLNa3-5 P637Q revealed that this mutation causes only minor structural changes close to mutation site. These changes were observed to significantly affect FLNa's ability to transmit cellular force and to interact with its binding partner. The performed steered molecular dynamics simulations showed that significantly lower forces are needed to split domains 4 and 5 in FLNA-MVD than with wild-type FLNa. The P637Q mutation was also observed to interfere with FLNa's interactions with the protein tyrosine phosphatase PTPN12. Our results provide a crucial step toward understanding the molecular bases behind FLNA-MVD, which is critical for the development of drug-based therapeutics.

Pathologic Oxidation of PTPN12 Underlies ABL1 Phosphorylation in Hereditary Leiomyomatosis and Renal Cell Carcinoma.

: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germline inactivating alterations in fumarate hydratase (FH) cause HLRCC and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH-/- PRCC cells have increased activation of ABL1, which promotes tumor growth, but how ABL1 is activated remains unclear. Given that oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity, inactivation of an ABL-directed PTP by ROS might account for ABL1 activation in this malignancy. Our group previously developed "q-oxPTPome," a method that globally monitors the oxidation of classical PTPs. In this study, we present a refined q-oxPTPome, increasing its sensitivity by >10×. Applying q-oxPTPome to FH-deficient cell models showed that multiple PTPs were either highly oxidized (including PTPN12) or overexpressed. Highly oxidized PTPs were those with relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells was reversible, although nearly 40% of PTPN13 was irreversibly oxidized to the sulfonic acid state. Using substrate-trapping mutants, we mapped PTPs to their putative substrates and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identified PTPN12 as the major ABL1 phosphatase, and overexpression of PTPN12 inhibited ABL1 phosphorylation and HLRCC cell growth. These results show that ROS-induced oxidation of PTPN12 accounts for ABL1 phosphorylation in HLRCC-associated PRCC, revealing a novel mechanism for inactivating a tumor suppressor gene product and establishing a direct link between pathologic PTP oxidation and neoplastic disease. SIGNIFICANCE: This work identifies a novel mechanism of activation of the oncogenic kinase ABL1 via ROS-induced, oxidation-mediated inactivation of cognate protein tyrosine phosphatases.