Academic literature on the topic 'PLEKHA1 [Pleckstrin homology domain containing, family A]'

Autosomal recessive intermediate Charcot Marie Tooth (CMT) disease type C is a very rarely-seen neurogenetic disorder. Homozygous or compound heterozygous mutation in the Pleckstrin homology domain-containing family G member 5 (PLEKHG5) gene on chromosome 1p36 was recently reported in patients with CMT. From the first description of the disease to date, almost 40 different variants associated with the PLEKHG5 gene were identified. Here, we present an adolescent girl who was thought initially to be myopathy because of progressive proximal muscle weakness. The electrophysiologic study revealed axonal sensory and motor neuropathy with some demyelinating features. She was diagnosed with autosomal recessive inheritance, intermediate CMT disease type C with a novel homozygous mutation in the PLEKHG5 gene in clinical exome sequencing as c.1600- 2A>G by next-generation sequencing. We describe here the novel mutation in the PLEKHG5 gene and the genotype-phenotype correlation.

Genetics intersects with environmental, cultural, and social factors in the development of addictive disorders. This study reports the feasibility of whole-exome sequencing of trios (subject and two family members) to discover potential genetic variants in the development of substance use disorders (SUD). Family trios were recruited from the National Addictions Management Service in Singapore during the 2016–2018 period. Recruited subjects had severe alcohol use disorder (AUD) or opioid use disorder (OUD), with nicotine dependence (ND) and a family history of addictive disorders. Demographic characteristics and severity of addiction were captured. Whole-exome sequencing (WES) and analysis were performed on salivary samples collected from the trios. WES revealed variants in several genes in each individual and disruptive protein mutations in most. Variants were identified in genes previously associated with SUDs, such as Pleckstrin homology domain-containing family M member 3 (PLEKHM3), coiled-coil serine-rich protein 1 (CCSER1), LIM and calponin homology domains-containing protein 1 (LIMCH1), dynein axonemal heavy chain 8 (DNAH8), and the taste receptor type 2 member 38 (TAS2R38) involved in the perception of bitterness. The feasibility study suggests that subjects with a severe addiction profile, polysubstance use, and family history of addiction may often harbor gene variants that may predispose them to SUDs. This study could serve as a model for future precision medicine-based personalized interventional strategies for behavioral addictions and SUDs and for the discovery of potentially pathogenic genetic variants.

Abstract Abstract 1284 Poster Board I-306 Recent SNP array analyses of B-acute lymphoblastic leukemia (B-ALL) have identified disruptions of the gene encoding the B-cell specific transcription factor Pax5 as one of the most common genomic lesions in this disease (> 30%); it being hemizygously deleted, mutated or involved in translocations. Pax5 translocates and forms fusion products with at least 12 different partners including C20orf112, leading to a chimeric Pax5/C20orf112 (Pax5/C20s) protein. Pax5 fusion products act as dominant negatives, competing for promoter binding sites with wild type (wt) Pax5 and thereby deregulating expression of target genes. In order to elucidate the molecular effects of fusion products involving the Pax5 gene, we performed a global gene expression analysis in the Nalm-6 B-ALL cell line. The cells were transfected with MSCV expression plasmids containing either empty vector, wild type Pax5 or a short fusion product of Pax5 and C20orf112 (Pax5/C20s), each containing IRES sequences for co-expression of GFP. Overexpression of Pax5 and Pax5/C20s was confirmed by western blot and quantitative RT PCR. RNA was extracted from cells sorted by FACS for GFP and processed for hybridization on Affymetrix HG-U133 plus 2 gene expression microarrays. Candidate genes were validated with RT real time PCR. Among the most differentially downregulated genes by the Pax5/C20s fusion product were candidate genes such as pleckstrin homology domain containing, family A member 2 (PLEKHA2) (12.64-fold), B-cell associated transcription factors POU class 2 associating factor 1 (POU2AF1) (4.4-fold) and transcription factor 3 (TCF3, E2A) (3.9-fold). Another intriguing observation was the downregulation of a group of genes associated with signaling through the pre-B cell receptor such as phosphoinositide-3-kinase adaptor protein 1 (BCAP) (3.35 fold), immunoglobulin heavy locus (IGH) (2.8 fold), pre-B lymphocyte genes -3 and -1 (VPREB3, VPREB1) (2.6-fold and 1.75-fold, respectively), spleen tyrosine kinase (SYK) (1.6 fold) and B-cell linker (SLP65, BLNK) (1.5-fold) by the Pax5/C20s fusion product. For stable expression and growth curves, Nalm6, 697, Kasumi2, RCH-ACV, SEM, HPB-Null, BV173 and BEL1 B-lymphoblastic cell lines were infected with retroviruses expressing the above mentioned retroviral expression constructs. We noted that forced expression of the PAX5/C20s fusion product inhibited growth in cell lines, which had functional pre-B cell receptor signaling. In contrast, the fusion gene either did not affect or enhanced growth of B-ALL cell lines, in which expression of a functional pre-B cell receptor was missing. Of note, Pax5 wt caused growth inhibition in B-ALL cell lines lacking functional pre-B cell receptor signaling. In cells with functional pre-B cell signaling, the response to engagement of the receptor as measured by calcium flux assay was diminished by overexpression of the Pax5/C20s fusion product as compared to empty vector control or PAX5 wt. These results suggest that the mechanisms of leukemogenesis of Pax5/C20s in ALL cells may be dependent on the functionality of the pre-B cell receptor pathway. This could be of great therapeutic value as it would potentially allow ALL cells to be divided into two different subtypes depending on pre-B cell receptor functionality and possibly identify the pre-B cell receptor pathway as a new therapeutic target. Disclosures No relevant conflicts of interest to declare.

Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain–dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

IntroductionA remarkable event that takes place during platelet activation is the reorganization that occurs when platelets adhere and spread on exposed collagen fibrils or become activated in the circulation by agonists, such as thrombin or adenosine diphosphate (ADP). In response to either stimulus, the shape of the platelet changes from a smooth disc to an irregular form with multiple, finger-like projections. This transformation is due to cytoskeletal rearrangements within the platelet. The platelet cytoskeleton is an intricately woven network1 arranged in three major structures: a cytoplasmic actin network, a rim of membrane-associated cytoskeleton, and a marginal band consisting of a microtubule coil. Together, these lend support to the platelet plasma membrane and give shape to both resting and activated platelets.At several levels, phosphoinositides are involved in the regulation of the platelet cytoskeleton. Actin binding, capping, and severing proteins are regulated by binding to phosphatidylinositol 4,5-bisphosphate (PIP2). The action of specific phosphoinositide kinases and phosphatases, leading to the regulation of levels of D3- and D4-containing phosphoinositides, has a profound impact on actin assembly. For example, synthesis of D3-containing phosphoinositides by phosphoinositide 3-kinases (PI3Ks) can lead to cortical actin assembly and the formation of lamellipodia downstream of stimulation by growth factor receptors, insulin receptors, and G protein-coupled receptors.2-5 There is increasing evidence that other lipid kinases also regulate cytoskeletal reorganization. Phosphatidylinositol 4-P 5-kinase enzymes, acting downstream of Rho family GTPases, have been shown to stimulate actin assembly.6 Because these areas have been covered in other articles,7,8 this review will, instead, concentrate on the role of pleckstrin and pleckstrin homology (PH) domains in the regulation of the actin cytoskeleton.Pleckstrin homology (PH) domains are the most wellrecognized phosphoinositide-binding protein motifs, comprising “modules” within more than 100 signaling proteins, and are used to mediate intermolecular interactions. The threedimensional structures of all PH domains studied to date are virtually superimposable, despite divergence in their amino acid sequence.9-17 The basic PH domain structure is composed of a β “sandwich,” capped at one end by a carboxyl-terminal α-helix, and all PH domains exhibit a striking polarity in their distribution of surface charge (Fig. 1). Based on the similarity of the structure of the NH2-terminal PH domain of pleckstrin to that of the retinol-binding protein, which was known to bind lipids, Harlan and coworkers tested PH domains and demonstrated that they bind to phosphoinositides.18 Since then, a number of laboratories, including our own, have published reports showing that the binding of PH domains to phosphoinositides can regulate protein function.4,19-22 It is now accepted that PH domains serve to localize their molecules into membrane structures by binding to phosphoinositides;18,23 though some PH domains may interact with other targets, such as the βγ subunits of heterotrimeric G proteins (Gβγ)24-27 or protein kinase C (PKC).28-30 The structure of several PH domains complexed to inositol trisphosphate (IP3) has been solved,11,13 confirming a physical interaction between the inositol phosphate headgroup and the positively charged face of the PH domain. For example, the association of the PH domain of PLCδ with IP3 is shown in Figure 1. Pleckstrin is a 43-kDa hematopoietic protein that contains the amino- and carboxyl- termini of the two prototypic PH domains (Fig. 2). Pleckstrin was first described as a major substrate for PKC in platelets and leukocytes, and its phosphorylation has long been used as a marker for platelet activation. Though its function in vivo remains unclear, expressed pleckstrin can affect PIP2-based signaling mediated by phospholipase C, PI3K, and inositol phosphatases.31-33 Ser113, Thr114, and Ser117, the three residues phosphorylated by PKC, lie adjacent to, but not within, the amino-terminal PH domain. Phosphorylation at these sites has been shown to regulate the function of this PH domain.34 Recently, a third functional motif has been described within pleckstrin.35 This motif is termed the DEP domain after the first three proteins known to possess this sequence (disheveled, Egl-10, and pleckstrin).

Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a ∼150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.

ABSTRACT Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.

碩士
國立陽明大學
生命科學系暨基因體科學研究所
103
Age-related macular degeneration (AMD) is a multifactorial disease for visual impairment in the senior population in developed countries. Clinical manifestations of AMD include the extracellular deposits of oxidized proteins and lipids within the retinal pigment epithelium (RPE). During the visual cycle, RPE supports the photoreceptor cells for the regeneration of visual pigments and breakdown of byproducts. Thus, dysfunction of RPE may result in metabolic burden to the photoreceptor cells. Although the exact cause of AMD is not clear, many studies have indicated that aging, oxidative stress, light damage and genetic factors may play significantly pathogenic roles. Both family and case-control studies revealed that genetic variants at 1q31 and 10q26 are the major genetic contributors. Further functional studies support that complement factor H (CFH) is the main player on chromosome 1q31, while the susceptibility gene on 10q26 remains to be elucidated. The aims of this study are (1) to reconstruct the risk haplotypes at 10q26 in exudative AMD, the prominent form of AMD in Asians strongly associated with the 10q26 variants, and (2) to investigate the role of candidate genes in this region during oxidative stress. Based on meta-analysis of GWAS data from studies of AMD, it was found that the risk locus extends more to the proximal region on 10q26 rather than to the distal region. In addition, due to no consensus on the functional roles for the other two candidates, ARMS2 and HTRA1, we therefore focused on PLEKHA1 as the candidate to investigate its possible role in AMD. So far, we have found that the two major isoforms of PLEKHA1 expressed differently in various tissues and the treatments of hydrogen peroxide, which mimics oxidative stress, induced translocations of PLEKHA1 isoform 1 to the plasma membrane but not isoform 2 in ARPE-19 cells. We further investigated the response of the two isoforms of PLEKHA1 under light exposure in the presence of N-retinyl-N-retinylidene ethanolamine (A2E), an autofluorescent pigment that accumulates in RPE cells in aging and some retinal disorders, which can induce generation of reactive oxygen species and cause serious toxicity to RPE cells. Similarly, A2E oxidative stress could induced translocations of PLEKHA1 isoform 1 to the plasma membrane in ARPE-19 cells. Induced phosphorylation of Akt (v-akt murine thymoma viral oncogen) in ARPE-19 cells under hydrogen peroxide or A2E oxidative stress was observed, but decreased level of phosphorylated Akt were observed in cells overexpressing PLEKHA1 isoform 1 rather than isoform 2. Both hydrogen peroxide and A2E oxidative stress induced cell death in ARPE-19 cells, but overexpressing PLEKHA1 isoform 1 slightly decrease resistance to oxidative stress whereas overexpression of PLEKHA1 isoform 2 slightly increase resistance to oxidative stress. To sum up, PLEKHA1 may play a role in cell survival under oxidative stress, thus it is a putative pathological cause for AMD.