Structural And Functional Characterization Of Phosphatidylethanolamine Binding Protein 4 (PEBP4) And Its Association With Type 2 Diabetes

Chapter 1 IntroductionDiabetes is a serious threat to human health and is becoming a global epidemic and more than 90% cases are type 2 diabetes(T2D). Due to a variety of factors, including genetic and environmental factors, T2 D is considered a multifactorial disease.In this study, we collected a family pedigree with a T2 D typical of dominant autosomal heredity. We then performed the whole genome exome sequencing and identified a nonsense mutation in the gene encoding phosphatidylethanolamine binding protein 4(PEBP4) in all affected members. PEBP4 has been previously demonstrated to regulate ERK1/2, Akt and ERα. To elucidate if and how PEBP4 is involved in onset of T2 D, we first use performed sequence alignment of PEBP1-4 as well as mouse PEBP4 and human PEBP4. The alignment suggests that PEBP4 is divergent from PEBP1-3 and PEBP4 contains a signal peptide sequence. To test if PEBP4 is secreted, we placed Myc epitope respectively to its amino-terminus and carboxyl-terminus to mask the signal peptide or keep it free. We then assessed the secretion of two versions of PEBP4, subcellular localization, and their effects on ERK and Akt activation. We also examined if the secreted PEBP4 is glycosylated by using mass spectrometry. Finally, we knocked out PEBP4 in mice and started to look into if this gene product is involved in pathogenesis of T2 D and metabolic syndrome.Chapter 2 Mutation of PEBP4 in the family of diabetesObjective: To unravel the molecular mechanism underlying T2 D linked with genetic trait.Methods: T2 D in a family with evident Mendel genetics were diagnosed by symptoms, physical signs and laboratory test. Peripheral blood from proband and siblings were collected for whole genome exome sequencing.Results: Proband’s father, uncle, three brother, a sister, four cousins proband himself were all diagnosed with T2 D in their forties. Proband’s three brother, a sister and proband himself were all found to contain a stop gain mutation in the PEBP4 gene, leading to encoding a premature protein with amino acids 1~172(instead of 224 amino acids in full length).Conclusions: Genetic predisposition is present in this T2 D family; Mutation of PEBP4 is associated with genetic T2 D.Chapter 3 PEBP4 is a secreted proteinObjective: To characterize structure and function of PEBP4 and its subcellular localization.Methods: Bioinfomatics was applied to align PEBP1-4, m PEBP4 and h PEBP. GFP or Myc epitope was tagged to human PEBP4 at the N-terminus or C-terminus(C’-/N’-GFP and Myc-PEBP4) and transfected into HEK293 T cells. Western blotting was used to detect PEBP4 in the cytosol and medium of HEK293 T cells. Confocal microscopy was used to observe PEBP4 subcellular localization.Results: Sequence alignment showed that PEBP1-3 are closely related, suggesting that they have similar functions, while PEBP4 has unique structure and function. Most carboxyl-terminally tagged h PEBP4 was secreted into cell culture media or showed a punctate distribution in the perinucleus(GFP fluorescence), while amino-terminally tagged recombinant protein was retained in the cytoplasm.Conclusions: PEBP4 is a secreted protein and free amino-terminal signal peptide is required for secretion.Chapter 4 PEBP4 is glycosylatedObjective: To determine glycosylation site2 on PEBP4.Methods: Using bioinformatics approach to predict glycosylation sites on h PEBP4 and m PEBP4; Site-directed mutagenesis of PEBP4 at R172(Stop codon). T171(to A), and E188(stop codon) were made and plasmid constructed and transfected into 293 T cells. Western blotting was used to detect mutant and wild-type PEBP4; Proteomic study, HILIC LC/MS or LC-MS/MS detected N-linked glycosylation.Results: Bioinformatics predicted an N-glycosylation site on h PEBP4(N169KT), and two(N140IT and N78IS) on m PEBP4. The prediction was verified by mass spectrometry and mutagenesis study of T171 A mutant.Conclusions: N169 KT on h PEBP4 and N140 IT and N78 IS on m PEBP4 are glycosylation sites.Chapter 5 The role of PEBP4 in activation of ERK and AktObjective: To investigate the effect of PEBP4 on the MAPK-ERK signaling pathway and the Akt signaling pathways.Methods: PEBP4-sh RNA was constructed and transfect into HEK 293 T cells, as compared with control sh RNA. C’-Myc-PEBP4 or N’-Myc-PEBP4 was transfected into HEK293 T cells. After treatment of the cells with or without EGF, cell extracts were blotted for p-ERK1/2, total ERK1/2, p-Akt, and total Akt.Results: PEBP4 sh RNA did not affect ERK activation although it silenced PEBP4. Overexpression of N’-Myc-PEBP4 suppressed ERK activation, but C’-Myc-PEBP4 did not have any effect. PEBP4 sh RNA suppressed Akt phosphorylation under basal conditions. C’-Myc-PEBP4 and N’-Myc-PEBP4 did not alter Akt phosphorylation, possibly attributable to constitutive activation of Akt in this cell lines.Conclusions: PEBP4 is not involved in the activation of ERK induced by EGF, but it is involved in the regulation of Akt signaling pathway.Chapter 6 Metabolic changes in PEBP4 knockout female miceObjective: To explore the effects of PEBP4 on energy metabolismMethods: PEBP4 knockout mice were generated, bred and screened. Ten female mice were grouped for homozygous and heterozygous deletion of PEBP4 and wild type littermates and subjected high fat diet for 3 months. Body weight, fasting glucose and glucose tolerance test(GTT) were measured every week over an 8-week period.Results: Body weight of female mice with PEBP4 knockout developed significantly quicker than that of wild type littermates. Knockout of PEBP4 caused an increase in fasting glucose and decreases in GTT and ITT in female mice fed with high fat diet.Conclusions: The PEBP4 gene plays a role in the regulation of glucose metabolism in female mice and its stop gain mutation may accounts for phenotypes of the hereditary T2 D family.