LncRNA SH3BP5-AS1 promotes hepatocellular carcinoma progression by sponging miR-6838-5p and activation of PTPN4.

BACKGROUND: Long noncoding RNAs (LncRNAs) have been demonstrated to have significant roles in the carcinogenesis of hepatocellular carcinoma (HCC). In this work, we sought to determine LncRNA SH3BP5-AS1's function and mechanism in the emergence of HCC. RESULTS: First, we discovered that the advanced tumor stage was strongly correlated with high levels of LncRNA SH3BP5-AS1 expression in HCC. MiR-6838-5p expression was down-regulated and inversely correlated with SH3BP5-AS1 expression. Additionally, overexpression of SH3BP5-AS1 boosted cell invasion, migration, and proliferation. The oncogenic effects of the inhibitor of miR-6838-5p were eliminated when PTPN4 was suppressed, following the identification of PTPN4 as a direct target of miR-6838-5p. In addition, SH3BP5-AS1 promoted cellular glycolysis via miR-6838-5p sponging and PTPN4 activation. Lastly, by directly interacting to the promoter of SH3BP5-AS1, HIF-1α could control the transcription of the gene. CONCLUSIONS: Our research suggests that SH3BP5-AS1 controls miR-6838-5p/PTPN4 in order to act as a new carcinogenic LncRNA during the growth of HCC cells. METHODS: The expression levels of SH3BP5-AS1, miR-6838-5p and PTPN4 were detected by qRT-PCR and Western blot. The effects of LncRNA SH3BP5-AS1/miR-6838-5p/PTPN4 on the proliferation, metastasis and glycolysis of HCC cells were clarified by experimental cellular functionality assays, cell derived xenograft and Glycolysis assay.

E3 Ubiquitin Ligase MARCH8 Promotes Pancreatic Cancer Growth and Metastasis by Activating STAT3 via Degradation of PTPN4.

OBJECTIVE: The role E3 ubiquitin ligase membrane-associated RING-CH 8 (MARCH8) has not been studied in pancreatic cancer. METHOD: Pancreatic cancer cell lines and the normal pancreatic cells were tested in vitro studies and male athymic nude mice were tested in vivo studies. Measuring cell viability by Cell Counting Kit-8 assay (CCK8), 5-ethynyl-2'- deoxyuridine (Edu) staining, and colony formation assay. Wound healing assay was implemented for cell migration and Transwell assay was performed for cell invasion to evaluate the histological status by hematoxylin and eosin staining and to detect the protein ubiquitination by ubiquitination assay. The protein expression was determined by immunohistochemistry staining and western blotting, and mRNA expression was measured by quantitative reverse transcription polymerase chain reaction. RESULT: The expression of MARCH8 was increased whereas PTPN4 was decreased in pancreatic cancer cells. Overexpression of MARCH8 promoted the growth, migration, and invasion of cells, and knockdown of PTPN4 had the similar effects both in vitro and in vivo. MARCH8 promoted PTPN4 protein degradation through ubiquitination. Moreover, PTPN4 suppressed the transcription activities of STAT3 by impairing the level of pSTAT3 (705), while inhibition of PTPN4 activated phosphorylation of STAT3. CONCLUSIONS: MARCH8 promoted pancreatic cancer growth and invasion through mediating the degradation of PTPN4 and activated the phosphorylation of STAT3.

MicroRNA-375 is a therapeutic target for castration-resistant prostate cancer through the PTPN4/STAT3 axis.

The functional role of microRNA-375 (miR-375) in the development of prostate cancer (PCa) remains controversial. Previously, we found that plasma exosomal miR-375 is significantly elevated in castration-resistant PCa (CRPC) patients compared with castration-sensitive PCa patients. Here, we aimed to determine how miR-375 modulates CRPC progression and thereafter to evaluate the therapeutic potential of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes loaded with miR-375 antisense oligonucleotides (e-375i). We used miRNA in situ hybridization technique to evaluate miR-375 expression in PCa tissues, gain- and loss-of-function experiments to determine miR-375 function, and bioinformatic methods, dual-luciferase reporter assay, qPCR, IHC and western blotting to determine and validate the target as well as the effects of miR-375 at the molecular level. Then, e-375i complexes were assessed for their antagonizing effects against miR-375. We found that the expression of miR-375 was elevated in PCa tissues and cancer exosomes, correlating with the Gleason score. Forced expression of miR-375 enhanced the expression of EMT markers and AR but suppressed apoptosis markers, leading to enhanced proliferation, migration, invasion, and enzalutamide resistance and decreased apoptosis of PCa cells. These effects could be reversed by miR-375 silencing. Mechanistically, miR-375 directly interfered with the expression of phosphatase nonreceptor type 4 (PTPN4), which in turn stabilized phosphorylated STAT3. Application of e-375i could inhibit miR-375, upregulate PTPN4 and downregulate p-STAT3, eventually repressing the growth of PCa. Collectively, we identified a novel miR-375 target, PTPN4, that functions upstream of STAT3, and targeting miR-375 may be an alternative therapeutic for PCa, especially for CRPC with high AR levels.

Progress in the mechanism of neuronal surface P antigen modulating hippocampal function and implications for autoimmune brain disease.

PURPOSE OF REVIEW: The aim of this study was to present a new regulation system in the hippocampus constituted by the neuronal surface P antigen (NSPA) and the tyrosine phosphatase PTPMEG/PTPN4, which provides mechanistic and therapeutic possibilities for cognitive dysfunction driven by antiribosomal P protein autoantibodies in patients with systemic lupus erythematosus (SLE). RECENT FINDINGS: Mice models lacking the function of NSPA as an E3 ubiquitin ligase show impaired glutamatergic synaptic plasticity, decreased levels of NMDAR at the postsynaptic density in hippocampus and memory deficits. The levels of PTPMEG/PTPN4 are increased due to lower ubiquitination and proteasomal degradation, resulting in dephosphorylation of tyrosines that control endocytosis in GluN2 NMDAR subunits. Adult hippocampal neurogenesis (AHN) that normally contributes to memory processes is also defective in the absence of NSPA. SUMMARY: NSPA function is crucial in memory processes controlling the stability of NMDAR at PSD through the ubiquitination of PTPMEG/PTPN4 and also through AHN. As anti-P autoantibodies reproduce the impairments of glutamatergic transmission, plasticity and memory performance seen in the absence of NSPA, it might be expected to perturb the NSPA/PTPMEG/PTPN4 pathway leading to hypofunction of NMDAR. This neuropathogenic mechanism contrasts with that of anti-NMDAR antibodies also involved in lupus cognitive dysfunction. Testing this hypothesis might open new therapeutic possibilities for cognitive dysfunction in SLE patients bearing anti-P autoantibodies.

Structural and biochemical analysis of the PTPN4 PDZ domain bound to the C-terminal tail of the human papillomavirus E6 oncoprotein.

High-risk genotypes of human papillomaviruses (HPVs) are directly implicated in various abnormalities associated with cellular hyperproliferation, including cervical cancer. E6 is one of two oncoproteins encoded in the HPV genome, which recruits diverse PSD-95/Dlg/ZO-1 (PDZ) domain-containing human proteins through its C-terminal PDZ-binding motif (PBM) to be degraded by means of the proteasome pathway. Among the three PDZ domain-containing protein tyrosine phosphatases, protein tyrosine phosphatase non-receptor type 3 (PTPN3) and PTPN13 were identified to be recognized by HPV E6 in a PBM-dependent manner. However, whether HPV E6 associates with PTPN4, which also has a PDZ domain and functions as an apoptosis regulator, remains undetermined. Herein, we present structural and biochemical evidence demonstrating the direct interaction between the PBM of HPV16 E6 and the PDZ domain of human PTPN4 for the first time. X-ray crystallographic structure determination and binding measurements using isothermal titration calorimetry demonstrated that hydrophobic interactions in which Leu158 of HPV16 E6 plays a key role and a network of intermolecular hydrogen bonds sustain the complex formation between PTPN4 PDZ and the PBM of HPV16 E6. In addition, it was verified that the corresponding motifs from several other high-risk HPV genotypes, including HPV18, HPV31, HPV33, and HPV45, bind to PTPN4 PDZ with comparable affinities, suggesting that PTPN4 is a common target of various pathogenic HPV genotypes.

Crocin induces ROS-mediated papillary thyroid cancer cell apoptosis by modulating the miR-34a-5p/PTPN4 axis in vitro.

miR-34a-5p has been reported to be upregulated and function as an oncogene in papillary thyroid cancer (PTC). Crocin, the major chemical constituent of saffron, has been demonstrated to possess anti-tumorigenic activity and decrease miR-34a-5p expression. Thus we hypothesized that crocin exerted anti-PCT effect by downregulating miR-34a-5p. Herein, the hypothetical mechanism underlying the anti-PCT effect of crocin was explored. Cell viability and apoptosis were assessed by CCK-8 and TUNEL assays, respectively. Reactive oxygen species (ROS) level, caspase-3 activity, and LDH release were measured using corresponding commercially available assay kits. Expression of miR-34a-5p and protein tyrosine phosphatase nonreceptor type 4 (PTPN4) was analyzed using qRT-PCR and western blot analyses. Interaction between miR-34a-5p and targets were predicted by Targetscan, starbase, miRDB, microT-CDS, and miRWalk and validated using luciferase reporter assay. Results showed that crocin inhibited the viability and miR-34a-5p expression in papillary thyroid cancer (PTC) cells in a dose-dependent manner. The Venn diagram showed that 10 overlapped targets of miR-34a-5p were identified, among which PTPN4 was the most significantly downregulated target gene in thyroid cancer tissues based on the heat map and bar plot from GSE33630 analysis. Luciferase reporter assay validated the direct interaction between miR-34a-5p and PTPN4. Crocin upregulated PTPN4 by decreasing miR-34a-5p expression in PTC cells. Crocin promoted apoptosis and increased caspase-3 activity and LDH release, which were reversed by ROS scavenger N-acetyl-L-cysteine (NAC), miR-34a overexpression, and PTPN4 silencing. To conclude, crocin promoted ROS-mediated apoptosis of PTC cells by modulating the miR-34a-5p/PTPN4 axis.

Neuronal surface P antigen (NSPA) modulates postsynaptic NMDAR stability through ubiquitination of tyrosine phosphatase PTPMEG.

BACKGROUND: Cognitive dysfunction (CD) is common among patients with the autoimmune disease systemic lupus erythematosus (SLE). Anti-ribosomal P autoantibodies associate with this dysfunction and have neuropathogenic effects that are mediated by cross-reacting with neuronal surface P antigen (NSPA) protein. Elucidating the function of NSPA can then reveal CD pathogenic mechanisms and treatment opportunities. In the brain, NSPA somehow contributes to glutamatergic NMDA receptor (NMDAR) activity in synaptic plasticity and memory. Here we analyze the consequences of NSPA absence in KO mice considering its structural features shared with E3 ubiquitin ligases and the crucial role of ubiquitination in synaptic plasticity. RESULTS: Electrophysiological studies revealed a decreased long-term potentiation in CA3-CA1 and medial perforant pathway-dentate gyrus (MPP-DG) hippocampal circuits, reflecting glutamatergic synaptic plasticity impairment in NSPA-KO mice. The hippocampal dentate gyrus of these mice showed a lower number of Arc-positive cells indicative of decreased synaptic activity and also showed proliferation defects of neural progenitors underlying less adult neurogenesis. All this translates into poor spatial and recognition memory when NSPA is absent. A cell-based assay demonstrated ubiquitination of NSPA as a property of RBR-type E3 ligases, while biochemical analysis of synaptic regions disclosed the tyrosine phosphatase PTPMEG as a potential substrate. Mice lacking NSPA have increased levels of PTPMEG due to its reduced ubiquitination and proteasomal degradation, which correlated with lower levels of GluN2A and GluN2B NMDAR subunits only at postsynaptic densities (PSDs), indicating selective trafficking of these proteins out of PSDs. As both GluN2A and GluN2B interact with PTPMEG, tyrosine (Tyr) dephosphorylation likely drives their endocytic removal from the PSD. Actually, immunoblot analysis showed reduced phosphorylation of the GluN2B endocytic signal Tyr1472 in NSPA-KO mice. CONCLUSIONS: NSPA contributes to hippocampal plasticity and memory processes ensuring appropriate levels of adult neurogenesis and PSD-located NMDAR. PTPMEG qualifies as NSPA ubiquitination substrate that regulates Tyr phosphorylation-dependent NMDAR stability at PSDs. The NSPA/PTPMEG pathway emerges as a new regulator of glutamatergic transmission and plasticity and may provide mechanistic clues and therapeutic opportunities for anti-P-mediated pathogenicity in SLE, a still unmet need.

miR-181c-5p Exacerbates Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Targeting PTPN4.

BACKGROUND: Activation of cell apoptosis is a major form of cell death during myocardial ischemia/reperfusion injury (I/RI). Therefore, examining ways to control cell apoptosis has important clinical significance for improving postischemic recovery. Clinical evidence demonstrated that miR-181c-5p was significantly upregulated in the early phase of myocardial infarction. However, whether or not miR-181c-5p mediates cardiac I/RI through cell apoptosis pathway is unknown. Thus, the present study is aimed at investigating the role and the possible mechanism of miR-181c-5p in apoptosis during I/R injury by using H9C2 cardiomyocytes. METHODS AND RESULTS: The rat origin H9C2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R, 6 hours hypoxia followed by 6 hours reoxygenation) to induce cell injury. The results showed that H/R significantly increased the expression of miR-181c-5p but not miR-181c-3p in H9C2 cells. In line with this, in an in vivo rat cardiac I/RI model, miR-181c-5p expression was also significantly increased. The overexpression of miR-181c-5p by its agomir transfection significantly aggravated H/R-induced cell injury (increased lactate dehydrogenase level and reduced cell viability) and exacerbated H/R-induced cell apoptosis (greater cleaved caspases 3 expression, Bax/Bcl-2 and more TUNEL-positive cells). In contrast, inhibition of miR-181c-5p in vitro had the opposite effect. By using computational prediction algorithms, protein tyrosine phosphatase nonreceptor type 4 (PTPN4) was predicted as a potential target gene of miR-181c-5p and was verified by the luciferase reporter assay. The overexpression of miR-181c-5p significantly attenuated the mRNA and protein expression of PTPN4 in H9C2 cardiomyocytes. Moreover, knockdown of PTPN4 significantly aggravated H/R-induced enhancement of LDH level, cleaved caspase 3 expression, and apoptotic cell death, which mimicked the proapoptotic effects of miR-181c-5p in H9C2 cardiomyocytes. CONCLUSIONS: These findings suggested that miR-181c-5p exacerbates H/R-induced cardiomyocyte injury and apoptosis via targeting PTPN4 and that miR-181c-5p/PTPN4 signaling may yield novel strategies to combat myocardial I/R injury.

Loss of PTPN4 activates STAT3 to promote the tumor growth in rectal cancer.

Colorectal cancer (CRC) is one of the most common types of malignant tumor. Many genetic factors have been proved to show high association with the occurrence and development of CRC and many mutations are detected in CRC. PTPN4/PTP-MEG1 is a widely expressed non-receptor protein tyrosine phosphatase. Over the past three decades, PTPN4 has been demonstrated in the literature to participate in many biological processes. In this study, we identified a nonsense mutation of PTPN4 with a mutation ratio of 90.90% from 1 case of rectal cancer, leading to loss of function in PTPN4 gene. Several somatic mutations occurred in 5/137 rectal cancer samples from The Cancer Genome Atlas Rectum Adenocarcinoma (TCGA READ) database. Interestingly, we found that PTPN4 negative cytoplasm staining was more prone to lymphatic metastasis (N = 50, P = 0.0153) and low expression of PTPN4 in rectal cancer was highly associated with poor prognosis. Overexpression of PTPN4 suppressed the cell growth, and moreover, the loss of PTPN4 accelerated cell growth and boosted clonogenicity of CRC cells. Furthermore, we revealed that the deletion of PTPN4 promoted the tumor formation of NCM460 cells in vivo. In terms of the molecular mechanism, we demonstrated that PTPN4 dephosphorylates pSTAT3 at the Tyr705 residue with a direct interaction and suppresses the transcriptional activity of STAT3. In summary, our study revealed a novel mechanism that the tumorigenesis of colorectal cancer might be caused by the loss of PTPN4 through activating STAT3, which will broaden the therapy strategy for anti-rectal cancer in the future.

Role of PTP/PTK trans activated insulin-like signalling pathway in regulation of grasshopper (Oedaleus asiaticus) development.

Protein tyrosine phosphatase (PTPs) and protein tyrosine kinase (PTKs) genes are responsible for the regulation of insect insulin-like pathway (ILP), cells growth, metabolism initiation, gene transcription and observing immune response. Signal transduction in insect cell is also associated with PTPs and PTKs. The grasshopper (Oedaleus asiaticus) 'Bey-Bienko' were treated with dsRNA of protein tyrosine non-receptor type 4 (PTPN4) and protein tyrosine kinase 5 (PTK5) along with control (water). Applying dsPTK5 treatments in 5th instar of Oedaleus asiaticus, significant reduction was recorded in body dry mass, growth rate and overall performance except survival rate. Whereas with PTPN4, no such significant impact on all of these growth parameters was recorded. Expression of genes in ILP 5th instar of Oedaleus asiaticus by the application of dsPTPN4 and dsPTK5 revealed that PTK, INSR (insulin receptor), IRS (insulin receptor substrate), PI3K (phosphoinositide 3-kinase), PDK (3-phosphoinositide-dependent protein kinase), Akt (protein kinase B) and FOXO (forkhead transcription factor) significantly expressed with downregulation except PTPN4, which remained non-significant. On the other hand, the phosphorylation level of ILP four proteins in O. asiaticus with the treatment of dsPTPN4 and dsPTK5 significantly affected P-IRS and P-FOXO, while P-INSR and P-AKT remained stable at the probability level of 5%. This indicated that the stress response in the O. asiaticus insulin-like signalling pathway (ILP) reduced. Regarding association of protective enzymatic activities, ROS (relative oxygen species), CAT (catalase) and PO (phenol oxidase) increased significantly with exposure to dsPTK5 as compared to dsPTPN4 and control, while exposure of 5th instar of O. asiaticus to dsPTPN4 treatment slightly raised CAT and PO activities with but significant contribution. No such significant effect on MFO and POD was seen using dsPTPN4 and dsPTK5. This showed that in the ILP of O. asiaticus, PTK5 was detrimental to growth, body mass and overall performance, which ultimately benefited insect detoxification with high-energy cost.

Neurodevelopmental phenotype caused by a de novo PTPN4 single nucleotide variant disrupting protein localization in neuronal dendritic spines.

Protein tyrosine phosphatase non-receptor type 4 (PTPN4) encodes non-receptor protein tyrosine phosphatase implicated in synaptic plasticity and innate immune response. The only report of PTPN4-associated disease described a neurodevelopmental disorder associated with a whole gene deletion. We describe a child with developmental delay, autistic features, hypotonia, increased immunoglobulin E and dental problems with a novel mosaic de novo variant in PTPN4 (hg19 chr2:g.120620188 T > C, NM_002830.3:p.[Leu72Ser]/c.215T>C) located in domain that controls protein subcellular distribution. Studies in mouse hippocampal neurons transfected with non-mutated or mutated human PTPN4 showed that despite their similar expression in neurons the mutated protein was absent from dendritic spines. Next, we studied patient's primary blood mononuclear cells' response to lipopolysaccharide stimulation and found no difference from control in phosphorylation of TBK1 and IRF3 (involved in Toll-like receptor 4 signaling) and induction of cytokines' messenger RNA. We conclude that the PTPN4 p.(Leu72Ser) variant is a likely cause of neurodevelopmental symptoms of our proband whereas its role in immune dysfunction requires further studies.

MicroRNA-208a Correlates Apoptosis and Oxidative Stress Induced by H2O2 through Protein Tyrosine Kinase/Phosphatase Balance in Cardiomyocytes.

MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, have been implicated as critical regulatory molecules in ischemia-/reperfusion-induced cardiac injury. In the present study, we report on the role of miR-208a in myocardial I/R injury and the underlying cardio-protective mechanism. The gain-of-function and loss-of-function were used to explore the effects of miR-208a on cardiac injury induced by H2O2 in cardiomyocytes. As predicted, knockdown of endogenous miR-208a significantly decreased the level of cellular reactive oxygen species (ROS) and reduced cardiomyocyte apoptosis. In addition, miR-208a overexpression increased the ROS level and attenuated cell apoptosis in cardiomyocytes. Furthermore, protein tyrosine phosphatase receptor type G (PTPRG) and protein tyrosine phosphatase, non-receptor type 4 (PTPN4), which participate in regulating the level of cellular protein tyrosine phosphorylation balance, were predicted and verified as potential miR-208a targets using bioinformatics and luciferase assay. In summary, this study demonstrated that miR-208a plays a critical protective role in ROS-induced cardiac apoptosis.

Structure-based prediction of protein- peptide binding regions using Random Forest.

Motivation: Protein-peptide interactions are one of the most important biological interactions and play crucial role in many diseases including cancer. Therefore, knowledge of these interactions provides invaluable insights into all cellular processes, functional mechanisms, and drug discovery. Protein-peptide interactions can be analyzed by studying the structures of protein-peptide complexes. However, only a small portion has known complex structures and experimental determination of protein-peptide interaction is costly and inefficient. Thus, predicting peptide-binding sites computationally will be useful to improve efficiency and cost effectiveness of experimental studies. Here, we established a machine learning method called SPRINT-Str (Structure-based prediction of protein-Peptide Residue-level Interaction) to use structural information for predicting protein-peptide binding residues. These predicted binding residues are then employed to infer the peptide-binding site by a clustering algorithm. Results: SPRINT-Str achieves robust and consistent results for prediction of protein-peptide binding regions in terms of residues and sites. Matthews' Correlation Coefficient (MCC) for 10-fold cross validation and independent test set are 0.27 and 0.293, respectively, as well as 0.775 and 0.782, respectively for area under the curve. The prediction outperforms other state-of-the-art methods, including our previously developed sequence-based method. A further spatial neighbor clustering of predicted binding residues leads to prediction of binding sites at 20-116% higher coverage than the next best method at all precision levels in the test set. The application of SPRINT-Str to protein binding with DNA, RNA and carbohydrate confirms the method's capability of separating peptide-binding sites from other functional sites. More importantly, similar performance in prediction of binding residues and sites is obtained when experimentally determined structures are replaced by unbound structures or quality model structures built from homologs, indicating its wide applicability. Availability and implementation: http://sparks-lab.org/server/SPRINT-Str. Contact: yangyd25@mail.sysu.edu.cn. Supplementary information: Supplementary data are available at Bioinformatics online.

Regulation of the Human Phosphatase PTPN4 by the inter-domain linker connecting the PDZ and the phosphatase domains.

Human protein tyrosine phosphatase non-receptor type 4 (PTPN4) has been shown to prevent cell death. The active form of human PTPN4 consists of two globular domains, a PDZ (PSD-95/Dlg/ZO-1) domain and a phosphatase domain, tethered by a flexible linker. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We previously demonstrated that the PDZ domain inhibits the phosphatase activity of PTPN4 and that the mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We demonstrate here that the linker connecting the PDZ domain and the phosphatase domain is involved in the regulation of the phosphatase activity in both PDZ-related inhibition and PDZ ligand-related activation events. We combined bioinformatics and kinetic studies to decipher the role of the linker in the PTPN4 activity. By comparing orthologous sequences, we identified a conserved patch of hydrophobic residues in the linker. We showed that mutations in this patch affect the regulation of the PTPN4 bidomain indicating that the PDZ-PDZ ligand regulation of PTPN4 is a linker-mediated mechanism. However, the mutations do not alter the binding of the PDZ ligand. This study strengthens the notion that inter-domain linker can be of functional importance in enzyme regulation of large multi-domain proteins.

Structure-based optimization of salt-bridge network across the complex interface of PTPN4 PDZ domain with its peptide ligands in neuroglioma.

The PTP non-receptor type 4 (PTPN4) is an important regulator protein in learning, spatial memory and cerebellar synaptic plasticity; targeting the PDZ domain of PTPN4 has become as attractive therapeutic strategy for human neuroglioma. Here, we systematically examined the complex crystal structures of PTPN4 PDZ domain with its known peptide ligands; a number of charged amino acid residues were identified in these ligands and in the peptide-binding pocket of PDZ domain, which can constitute a complicated salt-bridge network across the complex interface. Molecular dynamics (MD) simulations, binding free energy calculations and continuum model analysis revealed that the electrostatic effect plays a predominant role in domain-peptide binding, while other noncovalent interactions such as hydrogen bonds and hydrophobic forces are also responsible for the binding. The computational findings were then used to guide structure-based optimization of the interfacial salt-bridge network. Consequently, five peptides were rationally designed using the high-affinity binder Cyto8-RETEV (RETEV-COOH) as template, including four single-point mutants (i.e. Cyto8-mtxe0: RETEE-COOH, Cyto8-mtxd-1: RETDV-COOH, Cyto8-mtxd-3: RDTEV-COOH and Cyto8-mtxk-4: KETEV-COOH) and one double-point mutant (i.e. Cyto8-mtxd-1k-4: KETDV-COOH). Binding assays confirmed that three (Cyto8-mtxd-1, Cyto8-mtxk-4 and Cyto8-mtxd-1k-4) out of the five designed peptides exhibit moderately or considerably increased affinity as compared to the native peptide Cyto8-RETEV.

Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.

Phosphatase PTPN4 preferentially inhibits TRIF-dependent TLR4 pathway by dephosphorylating TRAM.

TLR4 recruits TRIF-related adaptor molecule (TRAM, also known as TICAM2) as a sorting adaptor to facilitate the interaction between TLR4 and TRIF and then initiate TRIF-dependent IRF3 activation. However, the mechanisms by which TRAM links downstream molecules are not fully elucidated. In this study, we show that TRAM undergoes tyrosine phosphorylation upon TLR4 activation and that is required for TLR4-induced IRF3 activation. Protein tyrosine phosphatase nonreceptor type 4 (PTPN4), a protein tyrosine phosphatase, inhibits tyrosine phosphorylation and subsequent cytoplasm translocation of TRAM, resulting in the disturbance of TRAM-TRIF interaction. Consequently, PTPN4 specifically inhibits TRIF-dependent IRF3 activation and IFN-ß production in TLR4 pathway. Therefore, our results provide new insight into the TLR4 pathway and identify PTPN4 as a specific inhibitor of TRIF-dependent TLR4 pathway. Targeting PTPN4 would be beneficial for the development of new strategy to control TLR4-associated diseases without unwanted side effects.

Strategies to interfere with PDZ-mediated interactions in neurons: What we can learn from the rabies virus.

PDZ (PSD-95/Dlg/ZO-1) domains play a major role in neuronal homeostasis in which they act as scaffold domains regulating cellular trafficking, self-association and catalytic activity of essential proteins such as kinases and phosphatases. Because of their central role in cell signaling, cellular PDZ-containing proteins are preferential targets of viruses to hijack cellular function to their advantage. Here, we describe how the viral G protein of the rabies virus specifically targets the PDZ domain of neuronal enzymes during viral infection. By disrupting the complexes formed by cellular enzymes and their ligands, the virus triggers drastic effect on cell signaling and commitment of the cell to either survival (virulent strains) or death (vaccinal strains). We provide structural and biological evidences that the viral proteins act as competitors endowed with specificity and affinity in an essential cellular process by mimicking PDZ binding motif of cellular partners. Disruption of critical endogenous protein-protein interactions by viral protein drastically alters intracellular protein trafficking and catalytic activity of cellular proteins that control cell homeostasis. This work opens up many perspectives to mimic viral sequences and developing innovative therapies to manipulate cellular homeostasis.

Regulation of the catalytic activity of the human phosphatase PTPN4 by its PDZ domain.

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cells death. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We demonstrate here that the PDZ domain inhibits the phosphatase activity of PTPN4. The mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We combined analytical ultracentrifugation, small angle X-ray scattering and NMR to understand how the PDZ domain controls PTPN4 activity. We show that the physiologically active PTPN4 two-domain, encompassing the PDZ and the phosphatase domains, adopts a predominant compact conformation in solution. The PDZ ligand binding restores the catalytic competence of PTPN4 disrupting the transient interdomain communication. This study strengthens the emerging notion that PDZ domains can act as regulators of enzyme activity and therefore are active players in the dynamic regulation of signaling pathways.

PTPN4 negatively regulates CrkI in human cell lines.

PTPN4 is a widely expressed non-receptor protein tyrosine phosphatase. Although its overexpression inhibits cell growth, the proteins with which it interacts to regulate cell growth are unknown. In this study, we identified CrkI as a PTPN4-interacting protein using a yeast two-hybrid, and confirmed this interaction using in vitro GST pull-down and co-immunoprecipitation and co-localization assays. We further determined the interactional regions as the SH3 domain of CrkI and the proline-rich region between amino acids 462 and 468 of PTPN4. Notably, overexpression of PTPN4 inhibits CrkI-mediated proliferation and wound healing of HEK293T cells, while knockdown of PTPN4 by siRNA in Hep3B cells enhances CrkI-mediated cell growth and motility. Moreover, our data show that ectopic expression of PTPN4 reduces the phosphorylation level of CrkI in HEK293T cells. These findings suggest that PTPN4 negatively regulates cell proliferation and motility through dephosphorylation of CrkI.