Thisproposedworkseekstodevelopasuiteofenablingtechnologiescapableofproducingsynthetic phosphoproteinswiththegoaloftransformingthefieldofhumanproteinsignalingfromonethatispurely observation?basedintoonethatbiosynthesizesdesignerproteinstoachieveacomprehensiveunderstandingof complexsignalingnetworks.Theimportanceofphosphorylationisemphasizedbythefactthat phosphorylatedproteinscontrolmostaspectsofnormalcellularhomeostasis.Aberrationsinprotein phosphorylationcandrivecancer,hypertension,diabetes,andneurodegenerativedisorders.Thus, understandingdifferentialpatternsofproteinphosphorylationindiseasestatesisofextremephysiological andclinicalinterest.Analysisofphosphorylatedaminoacidresidueshasbeenlimitedbyourinabilityto controlthesechemicalmodificationsduetoalackofphosphomimeticsthatfullyrecapitulatethechemistryof phosphorylatedresidues.Currentprogresstowardtheelucidationofphospho?signalingnetworksis hamperedbythelackofmethodstoproduceproteinscontainingspecificcombinationsofphosphorylated aminoacids.Inparticular,syntheticchemistryisinadequatefortotalphosphoproteinsynthesis,and conventionalbiologicalmethodsdonotcontrolphosphorylationlevels.Wehaverecentlydevelopedanew technology,albeitlimitedtophosphoserine(pSer),thatenablesthesynthesisofrecombinantphosphoproteins. Thistechnologydirectsphosphorylatedaminoacidsintotheirphysiologicallyrelevantpositionswithin proteinsyetourfunctionalunderstandingofproteinphosphorylationwillremainincompletewithoutaccess tophosphotyrosine(pTyr)andphosphothreonine(pThr)containingproteins.
SpecificAims :
In Aim1, wewill utilizemutagenesisandlaboratoryevolutiontoengineeranoptimizedtyrosylaminoacyl?tRNAsynthetasefor phosphotyrosine.
In Aim2, wewillprovideasolutiontothisproblembyengineeringanaminoacyl?tRNA synthetasethatcanchargeaphosphothreonineontoaspecialtRNAthatreadsadedicatedopencodon. Uniquetoourapproach,wewillalsoemployourgenomicallyrecodedE.colicellsinwhichopenstopcodons canbeconvertedintonewsensecodonsthatencodepThrandpTyrintopreciselocationsinrecombinant proteins.Significance:Theoveralloutcomeofourstudieswillbeanenablingtechnologyfortheexpressionof pTyrandpThrcontainingproteinsthatwillbroadlyenableresearchintodiseasemechanismsandcanbeused directlytodevelopnewtherapiesforhumandisease.Thiswillbethefirsttechnologyabletore?createhuman diseasenetworksthatare?difficult?or?impossible?toinfiltrate,andwillestablishtheparadigmfor addressingotherpost?translationalmodifications.Morebroadly,theproposedworkwillenablethere?design ofprogrammablesignalingnetworkscomprisingproteinswithnaturalandsyntheticnonstandardaminoacids capableofexpandingnetworksbeyondtheirnaturalfunctionsandofproducingnovelsyntheticpolymers withdiversechemistries.

Public Health Relevance

Human signaling networks involve large sets of proteins exhibiting highly complex patterns of phosphorylation. Since changes in phosphorylation levels occur at multiple sites (serine, threonine, tyrosine), understanding such networks at the systems and mechanistic levels remains an elusive and unmet goal. Here we seek to develop enabling technologies capable of encoding the nonstandard amino acids phosphothreonine and phosphotyrosine to produce synthetic phosphoproteins with the goal of transforming the field of human protein signaling from one that is purely observation-based into one that uses designer proteins to achieve a comprehensive understanding of complex signaling networks.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM125951-01
Application #
9423027
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Barski, Oleg
Project Start
2017-12-02
Project End
2021-11-30
Budget Start
2017-12-02
Budget End
2018-11-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520