Abstract

Lysosomescontrolasubstantialpartofcellularmetabolismbyactingasthemaincatabolichubofthecelland servingasaplatformfortheintegrationofnumeroussignalsthatmodulatecelldeath,growthandproliferation. Mostlysosomalfunctionsrelyonasetofmorethan50acidhydrolasesthatdegradeawidevarietyof macromolecules.Lysosomalenzymesaretraffickedtothelysosomeintwostages:transportofthenewly synthesizedproteinsfromtheendoplasmicreticulum(ER)totheGolgicomplex,andtheirsubsequent receptor-assistedtransferfromtheGolgitoendolysosomalcompartments.Howlysosomalenzymesare transportedfromtheERtotheGolgicomplexisunknownand,toourknowledge,thesimplemodelofabulk, unregulatedtransportationhasneverbeenquestioned.WehaveidentifiedtwocandidateERreceptors,CLN6 andCLN8,whosedeficiencyresultsinalteredmaturationoflysosomalenzymesandlysosomalstorage disorder-likediseases.WeproposetostudyhowCLN6andCLN8functioninthepathwayofmaturationof lysosomalenzymes.First,wewilltestthehypothesisthatCLN6andCLN8directlyinteractwithlysosomal enzymesandthatsuchinteractionisdisruptedbydisease-associatedmutationsoneitherCLN6/CLN8oron thesurfaceoflysosomalenzymes(Aim1).Second,wewillexaminethetraffickingandmaturationofnewly synthesizedlysosomalenzymestoidentifytheexactstepthatisdisruptedbyCLN6andCLN8deficiency.We willalsodefineCLN6andCLN8functionsinvivobycarryingoutdetailedtissue-specificanalysesoflysosomal compositioninCLN6-andCLN8-deficientmouselinesbyLC-MS/MS-basedproteomics.Tothisaim,wehave generatedaknock-inLamp1FLAGmouselinetoefficientlyisolatelysosomesfromthedesiredtissues(Aim2). Third,wewillidentifytheproteindomainsandmotifsthatareinvolvedinCLN6/CLN8interactionandthatdirect theirsortingacrossthecompartmentsoftheearlysecretorypathwayviaCOP-coatedvesicles(Aim3).Wewill accomplishourgoalswithamulti-disciplinaryapproachthatusesthetoolsofbiochemistry,molecularbiology, cellbiologyandmouseengineeringandwewillalsodevelopanewmethodofinvivolysosomeisolationfrom mousetissues.Ourresultsarelikelytohaveimportantconsequencesforourunderstandingofthe mechanismsgoverninglysosomalbiogenesisandofthemolecularpathogenesisofnumeroushuman diseases.Someoftheregulatorymechanismsweuncovermayserveinthefutureastargetsformodulating lysosomalbiogenesisindiseasesresultingfromimpairedlysosomalfunctionorinconditions,suchascertain typesofcancer,thatarecharacterizedbyaberrantorunrestrictedlysosomalactivation.

Public Health Relevance

Defectsinlysosomalhomeostasisandfunctionareimpliedinmanyhumanhealth-relatedconditionssuchas degenerativedisease,aging,disordersoftheimmunesystemandcancer.Wehavebeenstudyinglysosomal biogenesistoidentifytargetsandpathwaysthatmaybeleveragedtomodulatelysosomalfunctionsinhuman disease.Ouroverarchinggoalistoincreaseourmolecularunderstandingofthepathwayregulatingtheearly stepsofmaturationoflysosomalenzymes,whichservelysosomalbiogenesisasawhole,soastoenablethe communitytoexploitthepathwayfortherapeuticbenefit.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM127492-01A1
Application #
9660753
Study Section
Therapeutic Approaches to Genetic Diseases Study Section (TAG)
Program Officer
Ainsztein, Alexandra M
Project Start
2019-02-01
Project End
2023-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost

  Institution

Name
Baylor College of Medicine
Department
Genetics
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030