SODs and their metallochaperones in mitochondrial antioxidant defense

Transcription

1 ODs and their metallochaperones in mitochondrial antioxidant defense

2 The mitochondria as a factory for RO O 2? - O 2? - O 2? - O 2? - O 2? - O 2? - O 2? - O 2? -

3 Mn OD2

4 The metal containing superoxide dismutases.- + 2O 2 + 2H H 2 O 2 + O 2 OD1 = cytosolic, copper-requiring OD2 = mitochondria, manganese-requiring

5 Cu/Zn OD1 Highly conserved among eukaryotes Maturation of OD1: 1. Zinc insertion 2. Copper insertion 3. Disulfide bond formation Zn Cu Zn Cu OD1 Homodimer

6 1) O 2?- + Cu ++ Cu + + O 2 2) O 2? - + Cu + + 2H + Cu ++ + H 2 O 2 From Tainer et al., JMB 1982

7 OD1: Disulfide formation between C57 and C146 C146 C57 A rare cytosolic disulfide

8 Copper chaperones: escort proteins for metals From O Halloran and Culotta, J. Biol. Chem. 2000

9 Yeast cells lacking CC have no OD1 activity Wild type CC- Mn OD2 Cu/Zn OD1 Native gel assay for OD activity Western for OD1 protein

10 Mammalian OD1 has low activity without CC OD1 activity From Wong et al. Proc. Natl. Acad ci 2000

11 Copper activation of OD1 via CC From O Halloran and Culotta 2000

12 A fraction of OD1 and its copper chaperone CC localize to the mitochondria of yeast OD1 PM MIT0 CC MGE1 (mito) PGK1 (cyto) PM = post mitochondrial spnt; largely cytosol From turtz et al., JBC 2001

13 OD1 and CC localize to the intermembrane space of mitochondria PM Mito IM Mat OD1 CC PM = post-mitochondrial supernatant Mito = mitochondria IM = intermembrane space Mat = matrix turtz, et al. JBC 2001

14 OD1 in the mitochondria shows activity Wild type sod2? null PM mito PM mito OD2 activity OD1 activity OD1 polypeptide From turtz et al., JBC 2001

15 Mn OD2

16 What is the form of OD1 that enters mitochondria? H H H H H H Zn Zn Zn Zn H H H H Zn Cu Zn Cu Zn Cu Zn Cu H H

17 Apo, reduced OD1 is taken up most efficiently by mitochondria during in vitro import mitochondrial OD1 -OD1 E,E H E,E - E,Zn H E,Zn - Cu,Zn - Total OD1 Field, et al. JBC 2003

18 Mature OD1 Zn Cu Zn Cu Zn H H CC Zn H H Zn Cytosol Cu H H Mitochondrial IM H Zn Cu Zn Cu Zn H H CC Zn H H Zn H Mature OD1 Cu

19 OD1 appears to follow CC into mitochondria Mito IM pre-seq Yeast CC Cells expressing: Native CC pre-seq CC PM Mit IM Matrix PM Mit IM Matrix CC CC OD1 * * OD1 From turtz et al., JBC 2001

20 High levels of OD1 activity in cells with high mitochondrial CC Cells expressing: Native CC preseq-cc P M P M OD1 protein OD1 activity

21 How does CC affect mitochondrial accumulation of OD1? C C C C C C C Co-import? Mitochondrial retention?

22 CC helps to retain OD1 in the mitochondria No mitochondrial CC Total Retained Abundant mitochondrial CC Total Retained Mitochondrial OD1 C C C C Field, et al. JBC 2003

23 Disrupting OD1-CC interactions OD1 FG50,51EE (disrupts dimer interface) From Lamb et al., Nat. truc. Biol. 2001

24 OD1: P WT M F50E, G51E P M OD2 OD1 OD1 Disrupting the dimerization interface between OD1 and CC prevents mitochondrial accumulation of OD1 CC Field, et al. JBC 2003

25 tationary Phase urvival: A marker of chronological lifespan and oxidative damage Chronological life span in yeast: time a nondividing cell will survive in stationary phase Markers of oxidative damage and mitochondrial RO increase with time in stationary phase tationary phase survival is strongly influenced by anti-oxidant enzyme activity From the labs of V. Longo, J. Valentine, E. Gralla and T. Bilinski

26 sod1? yeast exhibit a shortened lifespan during stationary phase Day 2 WT sod1? turtz, et al. JBC 2001

27 IM OD1 prolongs lifespan during stationary phase OD2 OD1 native PM MITO IM-enriched PMMITO Day 2 Day 5 native OD1 IM-enriched OD1 turtz, et al. JBC 2001

28 Human OD1 mutations and familial AL (Lou Gehrigs disease)

29 Eukaryotic mitochondrial OD2 - a homotetramer D159 H163 From Borgstahl et al., Cell ) O 2?- + Mn +++ Mn ++ + O 2 2) O 2?- + Mn H + Mn H 2 O 2

30 OD2 must acquire its Mn in the mitochondria ynthesized in cytosol Activated in mitochondria

31 Mn insertion and mitochondrial import of OD1 appear coupled Newly synthesized OD2?

32 Possible mitochondrial transporters for manganese: The MCF (mito carrier family) transporters Intermembrane space N term C term Mitochondrial inner membrane Mitochondrial matrix

33 YGR257c = an unknown member of MCF family of mito transporters WT YGR257c? OD2 OD1 YGR257c deletion leads to virtual inactivation of OD2. OD1 activity increases probably due to compensatory mechanism.? -od2p

34 upplementation of Mn (but not other metals) fully restores OD2 activity WT ygr257c? WT ygr257c? WT ygr257c? +Mn +Cu +Fe WT ygr257c? OD2 OD1? -od2p

35 The MCF family of. cerevisiae Gene Loci Function Gene Loci Function MIR1 YJR077C phosphate AAC1 YMR056C ADP/ATP AAC2 YBL030C ADP/ATP AAC3 YBR085W ADP/ATP ARG11 YOR130C ornithine CAC1 YOR100C carnitine CTP1 YBR291C citrate ACR1 YJR095W dicarboxylate OAC1 YKL120W oxaloacetate DIC1 YLR348C dicarboxylate FLX1 YIL134W flavin LEU5 YHR002W acetylcoa MR3 YJL133W Mg++, Fe++? MR4 YKR052C Mg++, Fe++? RIM2 YBR192W Fe++, cysteine? YMC1 YPR058W? YMC2 YBR104W? PET8 YNL003C? YHM1 YDL198C? - YMR241W?? - YPR011C?? - YHR002W?? - YNL083W?? - YGR096W?? - YOR222W?? - YPL134C?? - YEL006W?? - YIL006W?? - YPR128C?? - YER053C?? - YER053C?? - YFR045W?? - YPR021C?? - YMR166C?? MTM1 YGR257C Mito transporter for Mn - YDL119C?? - YDR470C??

36 Mitochondrial manganese 100 Mitochondrial Mn (ng/mgs protein) 50 Wild type mtm1?

37

38 Human homologue to MTM1 Yeast Human Yeast Human Yeast Human Yeast Human Yeast Human Yeast Human ERMLAGAGVLTLI LTPMDVVRIR LQQQQMIP DCCDGAAEVPNAVGKMKTFTNV M+++GAG+V+TL +TP+DVV++RLQ Q+ P + K QQMVAGAGAVVTLF MTPLDVVKVRLQQRP ATELTTPRFWLYTKAL 6 8 GGQNLNNAKIF WEACFQELHCKN LKFNGTLEAFTKIA VEGI TLWRG 1 24 Q+ ++ C N + +F GTL+AF K I E G +LW G - - QPGKCLLYCNGVLEPLYLCPNGTRCATWFQDPTRFTGTLDAFVKIV RHEGTRTLWG 1 26 I LTLLMAI PANMVYFGYEYI RD-VPI ATYPTLNPLFCGAI ARVFAATIA PLELVK T L+ M + PA + YF+ Y T P+ GA+AR+ T ++ PLELV+ LPATLVMTVPATAI YFTAYDQLKAFLCGQLTDLYAPMVAGALARMGTVTVVPLELVR 1 86 TKLQI PRKTKTWMMVKDLLNETRQEMKMVGPRALFKGLEI TLWRDVPF AI YW 2 43 TKLQ+ + L + Q G R+L+ G T RDVPFA+YW + TKLQAQHVYRE LAVQAAVTQGGWRLWLGWGPTALRDVPFALYWFN 2 36 YELCKERLWLDTRFAKDANWVHFI NFAGCI GMIA AI CTHPFDVGKTRWQI MMNN 3 03 YEL K W L R KD V F +G I GM+AA T PFDV KT+ Q++ YELVK- - WL GLR-- PKDQTVGI- - FVAGGI GMVAATLTLPFDVVKTQRQMLGAV 2 90 DPK-GGNRRNMFKFLETIWRTEGLAALYTGLAARVIK I RPCAI MI YEI KKVF R + + L I G L+ G R+IK P CAI MI +YE K F EAVRVKPPRVDTWLLLRRIR AEGTRGLFAGFLPRI IK AAPCAI MI TYEFGKFF 34 8