Alteration of gene expression and protein solubility of the PI 5-phosphatase SHIP2 are correlated with Alzheimer's disease pathology progression.

A recent large genome-wide association study has identified EGFR (encoding the epidermal growth factor EGFR) as a new genetic risk factor for late-onset AD. SHIP2, encoded by INPPL1, is taking part in the signalling and interactome of several growth factor receptors, such as the EGFR. While INPPL1 has been identified as one of the most significant genes whose RNA expression correlates with cognitive decline, the potential alteration of SHIP2 expression and localization during the progression of AD remains largely unknown. Here we report that gene expression of both EGFR and INPPL1 was upregulated in AD brains. SHIP2 immunoreactivity was predominantly detected in plaque-associated astrocytes and dystrophic neurites and its increase was correlated with amyloid load in the brain of human AD and of 5xFAD transgenic mouse model of AD. While mRNA of INPPL1 was increased in AD, SHIP2 protein undergoes a significant solubility change being depleted from the soluble fraction of AD brain homogenates and co-enriched with EGFR in the insoluble fraction. Using FRET-based flow cytometry biosensor assay for tau-tau interaction, overexpression of SHIP2 significantly increased the FRET signal while siRNA-mediated downexpression of SHIP2 significantly decreased FRET signal. Genetic association analyses suggest that some variants in INPPL1 locus are associated with the level of CSF pTau. Our data support the hypothesis that SHIP2 is an intermediate key player of EGFR and AD pathology linking amyloid and tau pathologies in human AD.

Genetic Variants Associated with Hypertension Risk: Progress and Implications.

Background: Genetic variants causing diseases with hypertension as a secondary feature have previously been identified. Studies focussing on primary hypertension have utilised common and latterly rare genetic variants in attempts to elucidate the genetic contribution to the risk of primary hypertension. Summary: Using genome-wide association studies (GWASs), associations of hypertension with hundreds of common genetic variants have been reported, implicating thousands of genes. Individual variants have small effect sizes and cumulatively account for around 6% of genetic risk. The common variant signal is enriched for relevant tissues and physiological processes, while some variants are associated with traits expected to have secondary impacts on hypertension risk, such as fruit intake, BMI, or time watching television. Studies using rare variants obtained from exome sequence data have implicated a small number of genes for which impaired function has moderate effects on blood pressure and/or hypertension risk. Notably, genetic variants which impair elements of guanylate cyclase activation, stimulated by either natriuretic hormones or nitric oxide, increase hypertension risk. Conversely, variants impairing dopamine beta-hydroxylase or renin production are associated with lower blood pressure. Variants for which a definite effect can be designated remain cumulatively extremely rare and again make only a small contribution to overall genetic risk. Although these results are of interest, it is not clear that they provide radical new insights or identify drug targets which were not previously known. Nor does it seem that genetic testing could be useful in terms of quantifying disease risk or guiding treatment. Key Messages: Research has increased our knowledge about the relationship between naturally occurring genetic variation and risk of hypertension. Although some results serve to confirm our understanding of underlying physiology, their value in terms of potentially leading to practical advances in the management of hypertension appears questionable.

Analysis of Rare Variants in 470,000 Exome-Sequenced UK Biobank Participants Implicates Novel Genes Affecting Risk of Hypertension.

Introduction: A previous study of 200,000 exome-sequenced UK Biobank participants to test for association of rare coding variants with hypertension implicated two genes at exome-wide significance, DNMT3A and FES. A total of 42 genes had an uncorrected p value <0.001. These results were followed up in a larger sample of 470,000 exome-sequenced participants. Methods: Weighted burden analysis of rare coding variants in a new sample of 97,050 cases and 172,263 controls was carried out for these 42 genes. Those showing evidence for association were then analysed in the combined sample of 167,127 cases and 302,691 controls. Results: The association of DNMT3A and FES with hypertension was replicated in the new sample and they and the previously implicated gene NPR1, which codes for a membrane-bound guanylate cyclase, were all exome-wide significant in the combined sample. Also exome-wide significant as risk genes for hypertension were GUCY1A1, ASXL1, and SMAD6, while GUCY1B1 had a nominal p value of <0.0001. GUCY1A1 and GUCY1B1 code for subunits of a soluble guanylate cyclase. For two genes, DBH, which codes for dopamine beta hydroxylase, and INPPL1, rare coding variants predicted to impair gene function were protective against hypertension, again with exome-wide significance. Conclusion: The findings offer new insights into biological risk factors for hypertension which could be the subject of further investigation. In particular, genetic variants predicted to impair the function of either membrane-bound guanylate cyclase, activated by natriuretic peptides, or soluble guanylate cyclase, activated by nitric oxide, increase risk of hypertension. Conversely, variants impairing the function of dopamine beta hydroxylase, responsible for the synthesis of norepinephrine, reduce hypertension risk.

Structural Insights into the Binding Propensity of Human SHIP2 SH2 to Oncogenic CagA Isoforms from Helicobacter pylori.

SHIP2 is a multi-domain inositol 5-phosphatase binding to a variety of phosphotyrosine (pY)-containing proteins through its SH2 domain, so as to regulate various cell signaling pathways by modulating the phosphatidylinositol level in the plasma membrane. Unfavorably, Helicobacter pylori can hijack SHIP2 through the CagA protein to induce gastric cell carcinogenesis. To date, the interaction between SHIP2 and CagA was not analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Tyr-phosphorylated peptides from four EPIYA motifs (A/B/C/D) in CagA was studied using NMR spectroscopy. The results showed that EPIYA-C and -D bind to a similar interface of SHIP2-SH2, including a pY-binding pocket and a hydrophobic pocket, to achieve high affinity, while EPIYA-A and -B bind to a smaller interface of SHIP2-SH2 with weak affinity. By summarizing the interface and affinity of SHIP2-SH2 for CagA EPIYA-A/B/C/D, c-MET and FcgR2B ITIM, it was proposed that, potentially, SHIP2-SH2 has a selective preference for L > I > V for the aliphatic residues at the pY+3 position in its ligand. This study reveals the rule of the ligand sequence bound by SHIP2-SH2 and the mechanism by which CagA protein hijacks SHIP2, which will help design a peptide inhibitor against SHIP2-SH2.

An auxiliary binding interface of SHIP2-SH2 for Y292-phosphorylated FcγRIIB reveals diverse recognition mechanisms for tyrosine-phosphorylated receptors involved in different cell signaling pathways.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) plays an essential role in regulating phosphatidylinositol level in human cell, and is recruited to many phosphotyrosine (pY)-dependent signal transduction pathways by the SH2 domain. In immunity signaling, immunoreceptor FcγRIIB binds to SHIP2-SH2 via its Y292-phosphorylated immunoreceptor tyrosine-based inhibitory motif (ITIM) and transmits inhibitory signal, which regulates B cell and neuronal cell activity and is associated with immune diseases and Alzheimer's disease. To date, the interaction between SHIP2 and FcγRIIB has not been analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Y292-phosphorylated FcγRIIB-ITIM was analyzed using NMR spectroscopy. The results demonstrated that SHIP2-SH2 mainly utilizes two regions including a pY-binding pocket and a specificity pocket formed by ßD, ßE, and EF-loop, to bind with FcγRIIB-ITIM in high affinity. In addition to the two regions, the BG-loop of SHIP2-SH2 functions as an auxiliary interface enhancing affinity. By comparing the binding of SHIP2-SH2 with ligands from FcγRIIB and c-MET, a hepatocyte growth factor receptor associated with tumorigenesis, significant differences in interface and affinity were found, suggesting that SHIP2-SH2 applies diverse patterns for binding to different ligand proteins. Moreover, S49, S51, and R70 of SHIP2 were identified to mediate the binding of both FcγRIIB and c-MET, while R28 and Q107 were found to only participate in the binding of c-MET and FcγRIIB respectively. Taken together, this study reveals the diverse mechanisms of SHIP2-SH2 for recognizing different ligands, and provides important clues for selectively manipulating various signaling pathways and specific drug design.

Prenatal-onset INPPL1-related skeletal dysplasia in two unrelated families: Diagnosis and prediction of lethality.

This report describes two patients with INPPL1- related skeletal dysplasia diagnosed prenatally. A literature review is conducted to find out if high-lethality is associated with particular pathogenic variants in INPPL1 gene. Prediction of lethality in the prenatal setting has an impact on perinatal management. Some frameshift variants in INPLL1 gene are uniquely observed in lethal cases; however, more patients are needed to confirm the correlation.

Tribbles Pseudokinase 2 (TRIB2) Regulates Expression of Binding Partners in Bovine Granulosa Cells.

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5'-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.

Expression, purification and characterization of the RhoA-binding domain of human SHIP2 in E.coli.

Human SH2-containing inositol 5-phosphatase 2 (SHIP2) is a multi-domain protein playing essential roles in various physiological and pathological processes. In cell polarization and migration, SHIP2 serves as a RhoA effector for manipulating the level of phosphatidylinositol 3,4,5-trisphosphate. The domain between SH2 and a potential PH-R domain of SHIP2 was suggested to bind with GTP-bound form of RhoA. However, the structure of this RhoA-binding domain (RBD) of SHIP2 and the mechanism for its binding with RhoA remain unknown. In this study, SHIP2118-298 and SHIP2176-298, two truncated proteins harboring the RBD were designed, expressed, and purified successfully in E. coli. Unexpectedly, both SHIP2118-298 and SHIP2176-298 were determined to exist as homo-dimers in solution by multi-angle light scattering. Circular dichroism spectra indicated that both proteins predominantly consisted of α-helix structure. Moreover, in pull-down experiments, both proteins could bind with GTP-bound RhoA and RhoAQ63L, a mutant mimicing the state of GTP-bound RhoA. Importantly, in silico analysis showed that the shorter truncation, SHIP2176-298, contained all ordered residues between the SH2 and the PH-R domain, and matched the RhoA effector motif 1 of PKN1 well in sequence alignment, suggesting that SHIP2176-298 is sufficient for further studies on the structure and RhoA binding of SHIP2. This work shortens and confirms the main region of SHIP2 interacting with RhoA, provides the method for sample preparation, and presents preliminary information for SHIP2-RBD structure, which will facilitate the comprehensive understanding of the structure and function of SHIP2.

Met-Controlled Allosteric Module of Neural Generation as A New Therapeutic Target in Rodent Brain Ischemia.

OBJECTIVE: To investigate a Met-controlled allosteric module (AM) of neural generation as a potential therapeutic target for brain ischemia. METHODS: We selected Markov clustering algorithm (MCL) to mine functional modules in the related target networks. According to the topological similarity, one functional module was predicted in the modules of baicalin (BA), jasminoidin (JA), cholic acid (CA), compared with I/R model modules. This functional module included three genes: Inppl1, Met and Dapk3 (IMD). By gene ontology enrichment analysis, biological process related to this functional module was obtained. This functional module participated in generation of neurons. Western blotting was applied to present the compound-dependent regulation of IMD. Co-immunoprecipitation was used to reveal the relationship among the three members. We used IF to determine the number of newborn neurons between compound treatment group and ischemia/reperfusion group. The expressions of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) were supposed to show the changing circumstances for neural generation under cerebral ischemia. RESULTS: Significant reduction in infarction volume and pathological changes were shown in the compound treatment groups compared with the I/R model group (P<0.05). Three nodes in one novel module of IMD were found to exert diverse compound-dependent ischemic-specific excitatory regulatory activities. An anti-ischemic excitatory allosteric module (AME) of generation of neurons (AME-GN) was validated successfully in vivo. Newborn neurons increased in BJC treatment group (P<0.05). The expression of VEGF and MMP-9 decreased in the compound treatment groups compared with the I/R model group (P<0.05). CONCLUSIONS: AME demonstrates effectiveness of our pioneering approach to the discovery of therapeutic target. The novel approach for AM discovery in an effort to identify therapeutic targets holds the promise of accelerating elucidation of underlying pharmacological mechanisms in cerebral ischemia.

Altered chondrocyte differentiation, matrix mineralization and MEK-Erk1/2 signaling in an INPPL1 catalytic knock-out mouse model of opsismodysplasia.

Opsismodysplasia (OPS) is a rare but severe autosomal recessive skeletal chondrodysplasia caused by inactivating mutations in the Inppl1/Ship2 gene. The molecular mechanism leading from Ship2 gene inactivation to OPS is currently unknown. Here, we used our Ship2Δ/Δ mouse expressing reduced amount of a catalytically-inactive SHIP2 protein and a previously reported SHIP2 inhibitor to investigate growth plate development and mineralization in vivo, ex vivo and in vitro. First, as observed in OPS patients, catalytic inactivation of SHIP2 in mouse leads to reduced body length, shortening of long bones, craniofacial dysmorphism, reduced height of the hyperthrophic chondrocyte zone and to defects in growth plate mineralization. Second, intrinsic Ship2Δ/Δ bone defects were sufficient to induce the characteristic OPS alterations in bone growth, histology and mineralization ex vivo. Third, expression of osteocalcin was significantly increased in SHIP2-inactivated chondrocyte cultures whereas production of mineralized nodules was markedly decreased. Targeting osteocalcin mRNA with a specific shRNA increased the production of mineralized nodules. Fourth, levels of p-MEK and p-Erk1/2 were significantly increased in SHIP2-inactivated chondrocytes in response to serum and IGF-1, but not to FGF2, as compared to control chondrocytes. Treatment of chondrocytes and bones in culture with a MEK inhibitor partially rescued the production of mineralized nodules, the size of the hypertrophic chondrocyte zone and bone growth, raising the possibility of a treatment that could partially reduce the phenotype of this severe condition. Altogether, our results indicate that Ship2Δ/Δ mice represent a relevant model for human OPS. They also highlight the important role of SHIP2 in chondrocytes during endochondral ossification and its different differentiation steps. Finally, we identified a role of osteocalcin in mineralized nodules production and for the MEK-Erk1/2 signaling pathway in the OPS phenotype.

Underexpression of INPPL1 is associated with aggressive clinicopathologic characteristics in papillary thyroid carcinoma.

PURPOSE: To study the relationship between INPPL1 gene and clinicopathologic characteristics of papillary thyroid carcinoma (PTC). PATIENTS AND METHODS: INPPL1 expression in PTCs was tested by quantitative real-time reverse transcription PCR. The Cancer Genome Atlas (TCGA) RNA-seq data and our mRNA data were used to analyze and reveal the relationship between INPPL1 and aggressive clinicopathologic characteristics of PTC. RESULTS: When compared to normal thyroid tissues, INPPL1 was significantly downregulated in PTC tissues, as revealed by our data and TCGA data. INPPL1 underexpression was remarkably related to aggressive clinicopathologic characteristics such as lymph node metastasis (LNM), histological type, tumor size, mulitifocality, and disease stage in TCGA data. Meanwhile, LNM was confirmed to be associated with underexpression of INPPL1 in our data. In addition, logistic analysis clearly showed that underexpression of INPPL1 was an independent factor for LNM in PTC. CONCLUSION: INPPL1 may be a novel tumor suppressor gene in PTC, which was significantly correlated with aggressive clinicopathologic characteristics, especially LNM.

Solution structure of SHIP2 SH2 domain and its interaction with a phosphotyrosine peptide from c-MET.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) binds with the Y1356-phosphorylated hepatocyte growth factor (HGF) receptor, c-MET, through its SH2 domain, which is essential for the role of SHIP2 in HGF-induced cell scattering and cell spreading. Previously, the experimental structure of the SH2 domain from SHIP2 (SHIP2-SH2) had not been reported, and its interaction with the Y1356-phosphorylated c-MET had not been investigated from a structural point of view. In this study, the solution structure of SHIP2-SH2 was determined by NMR spectroscopy, where it was found to adopt a typical SH2-domain fold that contains a positively-charged pocket for binding to phosphotyrosine (pY). The interaction between SHIP2-SH2 and a pY-containing peptide from c-MET (Y1356 phosphorylated) was investigated through NMR titrations. The results showed that the binding affinity of SHIP2-SH2 with the phosphopeptide is at low micromolar level, and the binding interface consists of the positively-charged pocket and its surrounding regions. Furthermore, R28, S49 and R70 were identified as key residues for the binding and may directly interact with the pY. Taken together, these findings provide structural insights into the binding of SHIP2-SH2 with the Y1356-phosphorylated c-MET, and lay a foundation for further studies of the interactions between SHIP2-SH2 and its various binding partners.

The lipid 5-phoshatase SHIP2 controls renal brush border ultrastructure and function by regulating the activation of ERM proteins.

The microvillus brush border on the renal proximal tubule epithelium allows the controlled reabsorption of solutes that are filtered through the glomerulus and thus participates in general body homeostasis. Here, using the lipid 5-phosphatase Ship2 global knockout mice, proximal tubule-specific Ship2 knockout mice, and a proximal tubule cell model in which SHIP2 is inactivated, we show that SHIP2 is a negative regulator of microvilli formation, thereby controlling solute reabsorption by the proximal tubule. We found increased PtdIns(4,5)P2 substrate and decreased PtdIns4P product when SHIP2 was inactivated, associated with hyperactivated ezrin/radixin/moesin proteins and increased Rho-GTP. Thus, inactivation of SHIP2 leads to increased microvilli formation and solute reabsorption by the renal proximal tubule. This may represent an innovative therapeutic target for renal Fanconi syndrome characterized by decreased reabsorption of solutes by this nephron segment.

miR-184 functions as an oncogenic regulator in hepatocellular carcinoma (HCC).

Dysregulation of miRNAs has been proved to play a key role in carcinogenesis or tumor progression. In hepatocellular carcinoma (HCC), a number of miRNAs was reported to be related to the occurrence and development of HCC. Especially, miRNA-122, a liver-specific miRNA, has been elaborated its role in HCC. However, these studies was not involved in the effect of miRNA-184 on HCC. In the present study, we aimed to detect the miRNA-184 expression in HCC tissues and further evaluate the in vitro effect of miR-184 inhibition in HCC cells HepG2. We found that miR-184 expression was significantly high in HCC tissues, but INPPL1 expression was obviously low. Subsequently, INPPL1 was identified as a target of miRNA-184 by bioinformatics and dual luciferase assay. Moreover, after transfected with anti-miR-184 in HepG2 cells, INPPL1 expression was significantly decreased both at mRNA and protein levels. Additionally, we also proved that miR-184 silencing inhibited cellular proliferation by over expressing INPPL1 and induced HepG2 apoptosis by caspase 3/7. Together, our result was shown that miR-184 might play a part in proliferation of HCC cells by INPPL1 loss and act as antiapoptotic factor in the development of HCC by inhibiting the activities of caspases 3/7. Therefore, further elucidation of miRNA-184 silencing is helpful for understanding the pathogenesis of HCC and devising new strategies for its prevention and therapy.

High expression of inositol polyphosphate phosphatase-like 1 associates with unfavorable survival in hepatocellular carcinoma.

Inositol polyphosphate phosphatase-like 1 (INPPL1), also known as SH2-containing inositol 5'-phosphatase 2 (SHIP2), has been suggested to act downstream of the PI3K/AKT pathway and play an important function in tumor development and progression. However, the associations between SHIP2 expression and the clinical features to determine its clinicopathologic significance in hepatocellular carcinoma (HCC) have not been investigated. In the present study, one-step quantitative PCR reverse transcription-polymerase chain reaction (qPCR) and immunohistochemistry (IHC) analysis with HCC tissue microarrays (TMA) were employed to evaluate the expression of SHIP2 in HCC. The results showed that SHIP2 expression in the mRNA and protein levels was significantly higher in HCC tissue than in corresponding non-cancerous tissue (p = 0.0014 and p < 0.001, respectively). The expression of SHIP2 protein in HCC was related to tumor differentiation, α-fetoprotein level, liver cirrhosis, and five-year survival rate (all p < 0.05). Kaplan-Meier method and log-rank test indicated that high expression of SHIP2 (p = 0.017) and tumor differentiation (p = 0.036) showed significant correlations with poor prognosis of HCC patients. The data indicate that SHIP2 expression is correlated with significant characteristics of HCC, and it may be useful as an unfavorable prognostic factor in HCC.