Caspase 3-mediated cleavage of the vacuolar proton-pumping ATPase subunit ATP6V1B2
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1 Caspase 3-mediated cleavage of the vacuolar proton-pumping ATPase subunit ATP6V1B2 Usnish Majumdar Mentored by Prof. James Mandell, M.D., Ph.D. UVa Medical Center, Department of Pathology Supported by the U.Va. Summer Scholars Award *Artist s conception of a cell undergoing apoptosis. Cognition & David Ehlert.
2 Outline Caspases The brain A novel cleavage Future directions Crystal Structure: Caspase-3 with substrate Image from Protein Data Bank
3 What are caspases? Why study them?? Act as specific cellular scissors What s unknown?? Signal from Cell? Caspase-3 Activated Initiator Caspase Other Proteins???
4
5 Caspase cleavages in synapses Ethanol or saline treatment Purified mouse synapses ( synaptosomes ) Protein sequencing to discover substrate cleavages Saline Ethanol ~90 potential cleavages Of interest, ATP6V1B2 Sequencing Data Collaborator: Ken Victor, Pathology
6 B2 subunit of H + -V-ATPase Hypothesis: Cleavage of the ATP6V1B2 subunit of the vacuolar ATPase is mediated by caspase-3, and prevents loading and toxic release of transmitter from degenerating synapses Hilger and Jahn, 2011
7 B2 subunit cleavage Uncleaved B2: ~56 kd Major Cleavage Product: ~27 kd
8 Cleavage site identification Statistical prediction of caspase cut sites using Cascleave Generation of 3 B2 mutants, each with a mutated cleavage site Mahrus, Wells. Cell 134, , Sep. 5, kd 2.9 kd 4.5 kd 33 kd malramrgivngaapelpvptggpavgareqalavsrnylsqprltyktvsgvngplvildhvkfpryaeivhltlpdgtkrsgqvlevsgskavvqvfegtsgidakktsceftgdilrtpvsedmlgrvfngsgkpidrgpvvlaedfldimgqpinpqcriypeemiqtgisaidgmnsiargqkipifsaaglphneiaaqicrqaglvkkskdvvdyseenfaivfaamgvnmetarffksdfeengsmdnvclflnlandptieriitprlalttaeflayqcekhvlviltdmssyaealrevsaareevpgrrgfpgymytdlatiyeragrvegrngsitqipiltmpnddithpipdltgyitegqiyvdrqlhnrqiyppinvlpslsrlmksaigegmtrkdhadvsnqlyacyaigkdvqamkavvgeealtsddllyleflqkfernfiaqgpyenrtvfetldigwqllrifpkemlkripqstlsefyprdsakhdfld DFLD SAID DVVD DFLA SAIA DVVA
9 Cleavage site mutation Normal + Casp-3 (mutant) (mutant) (mutant) DVVA DFLA SAIA + Casp-3 + Casp-3 + Casp-3
10 Further observations Purified B2 synaptosomes Confirmation that B2 is cleaved Further physiological cleavage? No enzyme r Casp-3 r Casp-6 No enzyme r Casp-3 r Casp C-33.1kD kD DVVD* malramrgivngaapelpvptggpavgareqalavsrnylsqprltyktvsgvngplvildhvkfpryaeivhltlpdgtkrsgqvlevsgskavvqvfegtsgidakktsceftgdilrtpvsedmlgrvfngsgkpidrgpvvlaedfldimgqpinpqcriypeemiqtgisaidgmnsiargqkipifsaaglphneiaaqicrqaglvkkskdvvdyseenfaivfaamgvnmetarffksdfeengsmdnvclflnlandptieriitprlalttaeflayqcekhvlviltdmssyaealrevsaareevpgrrgfpgymytdlatiyeragrvegrngsitqipiltmpnddithpipdltgyitegqiyvdrqlhnrqiyppinvlpslsrlmksaigegmtrkdhadvsnqlyacyaigkdvqamkavvgeealtsddllyleflqkfernfiaqgpyenrtvfetldigwqllrifpkemlkripqstlsefyprdsakhdfld 16.1 kd 2.9 kd 4.5 kd 33 kd
11 Future: Functional Outcome of Caspase-3 Cleavage Assay of ATPase function Does cleavage destroy function? Can mutating the cleavage site rescue some of this loss? Studies of acidification Does cleavage raise the ph of the vesicle? Labeled lysosomes
12 Thanks to Mentor: James Mandell, M.D., PhD. Specialist: Dan Heffron Mass Spectrometry Collaboration: Ken Victor, PhD. The directors of the College Science Scholars Program: Professors Karen Schmidt, James Demas and Howard Epstein Many thanks go to the generous donors to the College Science Scholars program who make these summer research awards possible:charles Henry Leach, II, Foundation, Mr. Steve Van Besien; Entigence Corporation; Lois A. Fitton & W. Christopher Draper, Jr.; Sharon B. Parente & John W. Risner; The Jefferson Trust; Robert Atkinson; Dean s discretionary gifts
13 Questions?
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