Molecular mechanisms of PKA mutations underlying Cushing's syndrome

库欣综合征 PKA 突变的分子机制

• 批准号: 10377830
• 负责人: Mitchell Hamed Omar
• 金额: $ 5.09万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377830
• 关键词: A kinase anchoring protein; Adrenal Gland Adenoma; Adrenal Gland Hyperfunction; Adrenal Glands; Architecture; Binding; Biochemistry; Biology; Biosensor; Blood; CRISPR/Cas technology; Catalytic Domain; Cells; Chemicals; Chronic; Cognitive; Collaborations; Corticotropin; Cushing Syndrome; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Diabetes Mellitus; Diagnosis; Disease; Dissociation; Emotional; Endocrine Diagnostic Techniques; Endocrine System Diseases; Ensure; Environment; Enzymes; Face; Fluorescence Resonance Energy Transfer; Gene Proteins; Genes; Goals; Holoenzymes; Hormones; Hydrocortisone; Hypertension; Intervention; Island; Label; Lead; Link; Location; Macromolecular Complexes; Measurement; Microscopy; Modeling; Molecular; Mutation; Obesity; Pathologic; Pathway interactions; Patients; Pharmacology; Phosphorylation; Phosphotransferases; Physiological; Principal Investigator; Production; Proteins; Research; Research Personnel; Signal Transduction; Signaling Protein; Skin; Stress; Symptoms; Techniques; Testing; Thinness; Tissue Model; Training; Universities; Validation; Variant; Washington; Weight Gain; Work; base; body system; bone loss; experimental study; fluorophore; in vivo; innovation; live cell imaging; macromolecular assembly; mouse model; mutant; novel therapeutics; phosphoproteomics; prevent; protein complex; protein function; protein protein interaction; recruit; response; spatiotemporal; therapeutic candidate; tool

Project Summary    Chronically  high  levels  of  the  stress  hormone  cortisol  are  deleterious  to  many  organs  systems.  ACTH-­ independent  Cushing’s  syndrome  is  an  endocrine  disorder  wherein  the  adrenal  glands  constitutively  produce  excess  cortisol.  Symptoms  include  obesity,  thinning  of  the  skin,  cognitive  and  emotional  problems,  and  bone  loss. Recent studies have identified a mutation in the protein kinase A catalytic subunit (PKAc) in approximately  50%  of  these  cases.  This  mutation,  L205R,  is  on  the  face  of  PKAc  that  binds  to  its  regulatory  subunit,  and  is  predicted to disrupt holoenzyme formation.    Traditionally, the model for cyclic adenosine monophosphate (cAMP)-­protein kinase A (PKA) signaling has relied  on evidence using supraphysiological levels of cAMP stimulation, which leads to full dissociation of PKAc from  its regulatory subunit and from A-­kinase anchoring proteins (AKAPs). Recent evidence shows that physiological  levels  of  cAMP  do  not  dissociate  PKA  holoenzymes,  thereby  redefining  the  model  of  PKA  activation  in  cells.  Based  on  this  new  information,  the  proposed  experiments  will  test  if  Cushing’s  syndrome  mutations  in  PKAc  disrupt  localization  of  active  PKA  and  cause  ectopic  phosphorylation  of  substrates  and  downstream  cortisol  secretion. The hypothesis will be tested in two aims:    Aim 1: Is the spatiotemporal profile of PKAc L205R altered in Cushing’s syndrome? A combined strategy  of  CRISPR/Cas9  gene-­editing  and  live-­cell  imaging  with  photoactivatable  fluorophores  and  FRET-­based  biosensors will ascertain: 1) if mutant PKAc is recruited to AKAP-­signaling islands, 2) whether these Cushing’s  mutants are more mobile inside cells, and 3) if PKAc activity aberrantly accumulates at subcellular regions.     Aim 2: How does mutant PKAc cause excess cortisol production? I will use chemical biology techniques  combined  with  cortisol  measurement  in  NCI-­H295R  adrenal  cells  1)  to  determine  if  mislocalization  of  PKAc  activity is necessary and sufficient to boost cortisol release. Next, by combining miniTurbo proximity labeling with  phospho-­proteomics,  I  will  establish  2)  if  the  disease-­causing  L205R  PKAc  mutant  displays  altered  substrate  selectivity  that  adversely  impacts  downstream  signaling.  Additionally,  a  mouse  model  of  adrenal  PKAc  L205R  expression will be generated to evaluate candidate therapeutic intervention strategies resulting from this aim.    The  long-­term  goals  of  this  research  plan  are  to  elucidate  molecular  mechanisms  of  the  signaling  underlying  hypercortisolism in disease and to spur development of new therapeutic tools. The research will be conducted  at the University of Washington in the Department of Pharmacology. This environment provides excellent training  for  academic-­track  postdoctoral  researchers.  Training  benefits  include  strong  collaboration  within  and  among  departments,  approachable  principal  investigators  performing  innovative  work,  and  frequent  seminars  from  multiple departments featuring world experts in their respective fields.

项目总结