ABSTRACT
BACKGROUND: A targeted analysis of the 50kDa C-terminal fragment of insulin-response element binding protein-1 (IRE-BP1) activation of target genes through the insulin receptor substrate receptor/PI-3 kinase/Akt pathway has been demonstrated for the insulin growth factor-1 receptor. The broader effects of 50kDa C-terminal IRE-BP1 fragment over-expression on protein abundance in pancreatic islet beta cells have not been determined. RESULTS: Liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses of replicate lysates of pancreatic islets isolated from background strain animals and transgenic animals, overexpressing IRE-BP1 in pancreatic islet beta cells, demonstrated statistically significant increases in the expression of proteins involved in protein synthesis, endoplasmic reticulum (ER) stress and scaffolding proteins important for protein kinase C signaling; some of which were confirmed by immunoblot analyses. Bioinformatic analysis of protein expression network patterns suggested IRE-BP1 over-expression leads to protein expression patterns indicative of activation of functional protein networks utilized for protein post-translational modification, protein folding, and protein synthesis. Co-immunoprecipitation experiments demonstrate a novel interaction between two differentially regulated proteins receptor for activated protein kinase C (RACK1) and translationally controlled tumor protein (TCTP). CONCLUSIONS: Proteomic analysis of IRE-BP1 over-expression in pancreatic islet beta cells suggest IRE-BP1 (a) directly or indirectly through establishing hyperglycemia results in increased expression of ribosomal proteins and markers of ER stress and (b) leads to the enhanced and previously un-described interaction of RACK1 and TCTP. SIGNIFICANCE: This study identified C-terminal 50kDa domain of IRE-BP1 over-expression results in increased markers of ER-stress and a novel interaction between the scaffolding proteins RACK1 and TCTP.
Subject(s)
Biomarkers, Tumor/metabolism , Biomarkers/metabolism , Glucose/metabolism , Insulin-Secreting Cells/metabolism , Iron Regulatory Protein 1/metabolism , Islets of Langerhans/metabolism , Neuropeptides/metabolism , Animals , Endoplasmic Reticulum Stress/physiology , Hyperglycemia , Insulin/metabolism , Mice , Protein Kinase C/metabolism , Protein Processing, Post-Translational/physiology , Proteomics/methods , Receptors for Activated C Kinase , Response Elements/physiology , Tumor Protein, Translationally-Controlled 1ABSTRACT
The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG) growth conditions as compared to isoosmotic/normal glucose control (NG(â)) conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1) proteins involved in cell survival/cell signaling and (2) proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB), supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase), suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.
Subject(s)
Glucose/toxicity , Hyperglycemia/metabolism , Mesangial Cells/drug effects , Proteins/metabolism , Animals , Cell Line , Electrophoresis, Gel, Two-Dimensional , Homeostasis , Mesangial Cells/metabolism , Prohibitins , Protein Interaction Maps , Proteomics/methods , Rats , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time FactorsABSTRACT
Regulation of the endoplasmic reticulum (ER) stress-response pathway during the course of diabetes specifically in renal tubules is unclear. Since tubule cell dysfunction is critical to progression of diabetic nephropathy, this study analyzed markers of ER stress response and ER chaperones at different stages of diabetes and in different renal tubule subtypes of OVE26 type-1 diabetic mice. ER stress-responseinduced chaperones GRP78, GRP94, and protein disulfide isomerase (PDI) were increased in isolated cortical tubules of older diabetic mice, while PDI was decreased in tubules of young diabetic mice. Immunofluorescence staining of kidneys from older mice showed GRP78 and PDI upregulation in all cortical tubule segments, with substantial induction of PDI in distal tubules. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation was increased in cortical tubules of young diabetic mice, with no differences between older diabetic and control mice. Expression of ER stress-induced PERK inhibitor p58IPK was decreased and then increased in all tubule subtypes of young and older mice, respectively. Knockdown of PERK by small interfering RNA (siRNA) increased fibronectin secretion in cultured proximal tubule cells. Tubules of older diabetic mice had significantly more apoptotic cells, and ER stress-induced proapoptotic transcription factor C/EBP homologous protein (CHOP) was increased in proximal and distal tubules of diabetic mice and diabetic humans. CHOP induction in OVE26 mice was not altered by severity of proteinuria. Overexpression of CHOP in cultured proximal tubule cells increased expression of fibronectin. These findings demonstrate differential ER stress-response signaling in tubule subtypes of diabetic mice and implicate a role for PERK and CHOP in tubule cell matrix protein production.
Subject(s)
Diabetes Mellitus/pathology , Endoplasmic Reticulum Stress/physiology , Kidney Tubules, Distal/metabolism , Kidney Tubules, Proximal/metabolism , Transcription Factor CHOP/metabolism , eIF-2 Kinase/metabolism , Age Factors , Animals , Apoptosis/physiology , Cell Line , Disease Models, Animal , Endoplasmic Reticulum Chaperone BiP , Female , Fibronectins/metabolism , HSP40 Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/biosynthesis , Humans , Kidney Tubules, Distal/cytology , Kidney Tubules, Proximal/cytology , Membrane Glycoproteins/biosynthesis , Mice , Mice, Transgenic , Phosphorylation , Protein Disulfide-Isomerases/biosynthesis , Proteinuria/pathology , RNA Interference , RNA, Small Interfering/genetics , Transcription Factor CHOP/biosynthesis , Up-Regulation , eIF-2 Kinase/geneticsABSTRACT
Anacardic acid (AnAc; 2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with established anticancer activity in cell and animal models. The mechanisms by which AnAc inhibits cancer cell proliferation remain undefined. AnAc 24:1(omega5) was purified from geranium (Pelargonium x hortorum) and shown to inhibit the proliferation of estrogen receptor alpha (ERalpha)-positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with greater efficacy than ERalpha-negative primary human breast epithelial cells, MCF-10A normal breast epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1(omega5) inhibited cell cycle progression and induced apoptosis in a cell-specific manner. AnAc 24:1(omega5) inhibited estradiol (E(2))-induced estrogen response element (ERE) reporter activity and transcription of the endogenous E(2) target genes pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1(omega5) did not compete with E(2) for ERalpha or ERbeta binding, nor did AnAc 24:1(omega5) reduce ERalpha or ERbeta steady-state protein levels in MCF-7 cells; rather, AnAc 24:1(omega5) inhibited ER-ERE binding in vitro. Virtual screening with the molecular docking software Surflex evaluated AnAc 24:1(omega5) interaction with ERalpha ligand binding (LBD) and DNA binding (DBD) domains in conjunction with experimental validation. Molecular modeling revealed AnAc 24:1(omega5) interaction with the ERalpha DBD but not the LBD. Chromatin immunoprecipitation experiments revealed that AnAc 24:1(omega5) inhibited E(2)-ERalpha interaction with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc 24:1(omega5) inhibits cell proliferation, cell cycle progression, and apoptosis in an ER-dependent manner by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses.
Subject(s)
Anacardic Acids/pharmacology , Breast Neoplasms/pathology , Cell Proliferation/drug effects , DNA/metabolism , Estrogen Receptor alpha/antagonists & inhibitors , Transcription, Genetic/drug effects , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , DNA/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Genes, Reporter/drug effects , Humans , Protein Binding/drug effects , Transcriptional Activation/drug effects , Transfection , Tumor Cells, CulturedABSTRACT
Escuela Kleiniana
ABSTRACT
Escuela Kleiniana
ABSTRACT
Este libro explica el comportamiento propio de los niños pequeños a la luz de su desarrollo físico, intelectual y emocional y ofrece consejos útiles para la vida del hogar
ABSTRACT
Este libro explica el comportamiento propio de los niños pequeños a la luz de su desarrollo físico, intelectual y emocional y ofrece consejos útiles para la vida del hogar
