Endosomal phosphoinositides and human diseases

Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking. Seven distinct PIs can be synthesized by phosphorylation of the inositol ring of phosphatidylinositol (PtdIns), and their metabolism is accurately regulated by PI kinases and phosphatases. Two of the PIs, PtdIns3 P and PtdIns(3,5) P ₂, are present on intracellular endosomal compartments, and several studies suggest that they have a role in membrane remodeling and trafficking. We refer to them as 'endosomal PIs'. An increasing number of human genetic diseases including myopathy and neuropathies are associated to mutations in enzymes regulating the turnover of these endosomal PIs. The PtdIns3 P and PtdIns(3,5) P ₂ 3-phosphatase myotubularin gene is mutated in X-linked centronuclear myopathy, whereas its homologs MTMR2 and MTMR13 and the PtdIns(3,5) P ₂ 5-phosphatase SAC3/FIG4 are implicated in Charcot–Marie–Tooth peripheral neuropathies. Mutations in the gene encoding the PtdIns3 P 5-kinase PIP5K3/PIKfyve have been found in patients affected with François–Neetens fleck corneal dystrophy. This review presents the roles of the endosomal PIs and their regulators and proposes defects of membrane remodeling as a common pathological mechanism for the corresponding diseases.     

Diacylglycerol kinase gamma interacts with and activates beta2-chimaerin, a Rac-specific GAP, in response to epidermal growth factor

Diacylglycerol kinase (DGK)gamma was shown to act as an upstream suppressor of Rac1. Here we report that, in COS7 cells stimulated with epidermal growth factor (EGF), DGKgamma specifically interacts and co-localizes at the plasma membrane with beta2-chimaerin, a GTPase-activating protein (GAP) for Rac. Moreover, DGKgamma enhanced EGF-dependent translocation of beta2-chimaerin to the plasma membrane. Interestingly, DGKgamma markedly augmented EGF-dependent GAP activity of beta2-chimaerin through its catalytic action. These results indicate that DGKgamma is a novel regulator of beta2-chimaerin, and thus suggest that beta2-chimaerin is an effector molecule, linking DGKgamma functionally with Rac1.     

Discovery and pharmacological characterization of a novel small molecule inhibitor of phosphatidylinositol-5-phosphate 4-kinase,type II,beta

Phosphatidylinositol-5-phosphate 4-kinase, type II, beta (PIP5K2B) is linked to the pathogenesis of obesity, insulin resistance and diabetes. Here, we describe the identification of a novel pyrimidine-2,4-diamine PIP5K2B inhibitor, designated SAR088. The compound was identified by high-throughput screening and subsequently characterized in vitro and in vivo. SAR088 showed reasonable potency, selectivity and physicochemical properties in enzymatic and cellular assays. In vivo, SAR088 lowered blood glucose levels of obese and hyperglycemic male Zucker diabetic fatty rats treated for 3 weeks. Thus, SAR088 represents the first orally available and in vivo active PIP5K2B inhibitor and provides an excellent starting point for the development of potent and selective PIP5K2B inhibitors for the treatment of insulin resistance and diabetes.     

Cellular prostatic acid phosphatase,a PTEN-functional homologue in prostate epithelia,functions as a prostate-specific tumor suppressor

The inactivation of tumor suppressor genes (TSGs) plays a vital role in the progression of human cancers. Nevertheless, those ubiquitous TSGs have been shown with limited roles in various stages of diverse carcinogenesis. Investigation on identifying unique TSG, especially for early stage of carcinogenesis, is imperative. As such, the search for organ-specific TSGs has emerged as a major strategy in cancer research. Prostate cancer (PCa) has the highest incidence in solid tumors in US males. Cellular prostatic acid phosphatase (cPAcP) is a prostate-specific differentiation antigen. Despite intensive studies over the past several decades on PAcP as a PCa biomarker, the role of cPAcP as a PCa-specific tumor suppressor has only recently been emerged and validated. The mechanism underlying the pivotal role of cPAcP as a prostate-specific TSG is, in part, due to its function as a protein tyrosine phosphatase (PTP) as well as a phosphoinositide phosphatase (PIP), an apparent functional homologue to phosphatase and tensin homolog (PTEN) in PCa cells. This review is focused on discussing the function of this authentic prostate-specific tumor suppressor and the mechanism behind the loss of cPAcP expression leading to prostate carcinogenesis. We review other phosphatases' roles as TSGs which regulate oncogenic PI3K signaling in PCa and discuss the functional similarity between cPAcP and PTEN in prostate carcinogenesis.     

The TLR3, PI3K,survin, FasL,and Fas genes as major risk factors of occurrence and development of cervical cancer disease

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3–siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3–siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3–siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.     

GABA/progesterone-induced polyphosphoinositide (PPI) breakdown and its role in the acrosome reaction of guinea pig spermatozoain vitro

To investigate whether GABA/progesterone (P₄) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa²⁺ for 5.5 h and then labeled with [³²P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5×10⁷ cells/mL and exposed to 2 mmol/L Ca²⁺, 5 ?mol/L GABA, 10 ?mol/L P₄ and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermatozoa were treated with GABA, ³²P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa reached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation of AR was similar for the three agonists tested;their potency followed the order A23187＞progesterone≥GABA; (iii) GABA-induced PIP₂ hydrolysis and rise in PA and the AR were prevented by inclusion of 10 mmol/L neomycin; (iv) the loss of PIP2 labeling and the increase in PA labeling abolished when spermatozoa were exposed to EGTA or Ca²⁺ channel blocker. These results indicate that GABA or P₄^-induced PPI breakdown is an important and essential event in the series of changes to membrane fusion during the AR of guinea pig spermatozoa and this effect is mediated via calcium by activation of phosphatidylinositol-specific phospholipase C.     

A Phylogenetic Framework for the Aquaporin Family in Eukaryotes

A comprehensive evolutionary analysis of aquaporins, a family of intrinsic membrane proteins that function as water channels, was conducted to establish groups of homology (i.e., to identify orthologues and paralogues) within the family and to gain insights into the functional constraints acting on the structure of the aquaporin molecule structure. Aquaporins are present in all living organisms, and therefore, they provide an excellent opportunity to further our understanding of the broader biological significance of molecular evolution by gene duplication followed by functional and structural specialization. Based on the resulting phylogeny, the 153 channel proteins analyzed were classified into six major paralogous groups: (1) GLPs, or glycerol-transporting channel proteins, which include mammalian AQP3, AQP7, and AQP9, several nematode paralogues, a yeast paralogue, and Escherichia coli GLP; (2) AQPs, or aquaporins, which include metazoan AQP0, AQP1, AQP2, AQP4, AQP5, and AQP6; (3) PIPs, or plasma membrane intrinsic proteins of plants, which include PIP1 and PIP2; (4) TIPs, or tonoplast intrinsic proteins of plants, which include alphaTIP, gammaTIP, and deltaTIP; (5) NODs, or nodulins of plants; and (6) AQP8s, or metazoan aquaporin 8 proteins. Of these groups, AQPs, PIPs, and TIPs cluster together. According to the results, the capacity to transport glycerol shown by several members of the family was acquired only early in the history of the family. The new phylogeny reveals that several water channel proteins are misclassified and require reassignment, whereas several previously undetermined ones can now be classified with confidence. The deduced phylogenetic framework was used to characterize the molecular features of water channel proteins. Three motifs are common to all family members: AEF (Ala-Glu-Phe), which is located in the N-terminal domain; and two NPA (Asp-Pro-Ala) boxes, which are located in the center and C-terminal domains, respectively. Other residues are found to be conserved within the major groups but not among them. Overall, the PIP subfamily showed the least variation. In general, no radical amino acid replacements affecting tertiary structure were identified, with the exception of Ala-->Ser in the TIP subfamily. Constancy of rates of evolution was demonstrated within the different paralogues but rejected among several of them (GLP and NOD).     

Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family

Plasma membrane intrinsic proteins （PIPs） are a subfamily ofaquaporins that enable fast and controlled translocation of water across the membrane. In this study, we systematically identified and cloned ten PIP genes from rice. Based on the similarity of the amino acid sequences they encoded, these rice PIP genes were classified into two groups and designated as OsPIP1-1 to OsPIP1-3 and OsPIP2-1 to OsPIP2-7 following the nomenclature of PIP genes in maize. Quantitative RT-PCR analysis identified three root-specific and one leaf-specific OsPIP genes. Furthermore, the expression profile of each OsPIP gene in response to salt, drought and ABA treatment was examined in detail. Analysis on transgenic plants over-expressing of either OsPIP1 （OsPIP1-1） or OsPIP2 （OsPIP2-2） in wild-type Arabidopsis, showed enhanced tolerance to salt （100 mM of NaCl） and drought （200 mM ofmannitol）, but not to salt treatment of higher concentration （150 mM of NaCl）. Taken together, these data suggest a distinct role of each OsPIP gene in response to different stresses, and should add a new layer to the understanding of the physiological function of rice PIP genes.     

跨膜蛋白CD3ε与磷脂酰肌醇二磷酸在脂筏域体系中蛋白质-脂质相互作用的粗粒化模拟研究

细胞膜局部区域可形成富含饱和脂质、胆固醇、鞘脂的脂筏域作为其信号转导调控平台。传统实验手段在研究脂筏及其功能时受到系统复杂度高及区域结构瞬时性强等制约。近年来,分子动力学模拟技术为细胞膜的组织原则提供了重要的理论支撑,从简单的单一组分模型到多组分系统转变,最终形成了越来越多的细胞膜仿真模型。其中,粗粒化模拟由于其简化模型,可大副拓展模拟体系的复杂程度与模拟时间,在细胞膜以及蛋白质-脂质相互作用相关研究中得到了广泛应用。本文采用MARTINI粗粒化力场模拟,构建了一种含有阴离子脂质磷脂酰肌醇二磷酸(phosphatidylinositol diphosphate, PIP2)的混合膜体系。模拟结果表明,该体系在适当温度及饱和度条件下,能自发分层形成脂筏域;膜厚度、膜组分分布、膜组分流动性等多种参数均表明,脂筏结构形成且符合其结构特征;少量PIP2添加不影响分层特性且PIP2对脂筏具有显著亲和性。此外,利用该模型以跨膜信号蛋白CD3ε为例研究了脂筏域体系中蛋白质-脂质相互作用。结果表明,PIP2-CD3ε胞内区相互作用可能是脂筏招募CD3ε的驱动力,且该过程可受钙离子调控。本工作体现了粗粒化模拟在仿真膜相关研究中的巨大优势及良好应用前景。     

PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

Variants identified in genome‐wide association studies have implicated immune pathways in the development of Alzheimer’s disease (AD). Here, we investigated the mechanistic basis for protection from AD associated with PLCγ2 R522, a rare coding variant of the PLCG2 gene. We studied the variant''s role in macrophages and microglia of newly generated PLCG2‐R522‐expressing human induced pluripotent cell lines (hiPSC) and knockin mice, which exhibit normal endogenous PLCG2 expression. In all models, cells expressing the R522 mutation show a consistent non‐redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This manifests as enhanced release of cellular calcium ion stores in response to physiologically relevant stimuli like Fc‐receptor ligation or exposure to Aβ oligomers. Expression of the PLCγ2‐R522 variant resulted in increased stimulus‐dependent PIP₂ depletion and reduced basal PIP₂ levels in vivo. Furthermore, it was associated with impaired phagocytosis and enhanced endocytosis. PLCγ2 acts downstream of other AD‐related factors, such as TREM2 and CSF1R, and alterations in its activity directly impact cell function. The inherent druggability of enzymes such as PLCγ2 raises the prospect of PLCγ2 manipulation as a future therapeutic approach in AD.