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.
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.
| Barber, Karl W; Muir, Paul; Szeligowski, Richard V et al. (2018) Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions. Nat Biotechnol 36:638-644 |
| Barber, Karl W; Rinehart, Jesse (2018) The ABCs of PTMs. Nat Chem Biol 14:188-192 |
| Ma, Natalie Jing; Hemez, Colin F; Barber, Karl W et al. (2018) Organisms with alternative genetic codes resolve unassigned codons via mistranslation and ribosomal rescue. Elife 7: |
| Barber, Karl W; Miller, Chad J; Jun, Jay W et al. (2018) Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library. Biochemistry 57:4717-4725 |
