title: Deregulation of mitochondrial membrane potential by mitochondrial insertion of granzyme B and direct Hax-1 cleavage.
abstract: The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrB-treated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.
authors.affiliation: Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Han
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Goldstein
authors.forename: Wen
authors.initials: W
authors.lastname: Hou
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Froelich
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.forename: Hannah
authors.initials: H
authors.lastname: Rabinowich
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects; D045744:Cell Line, Tumor; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000081406:Peptidyl-Prolyl Isomerase F; C104759:HAX1 protein, human; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; C509213:GZMB protein, human; D053804:Granzymes; D021983:Cyclophilins; D013752:Tetracycline
references.citation: J Immunol. 1997 Mar 15;158(6):2736-44
references.pmid: 9058808
references.citation: Nat Rev Immunol. 2002 Jun;2(6):401-9
references.pmid: 12093006
references.citation: Curr Opin Immunol. 2003 Oct;15(5):553-9
references.pmid: 14499264
references.citation: Nature. 2005 Mar 31;434(7033):658-62
references.pmid: 15800627
references.citation: J Biol Chem. 2004 Nov 26;279(48):50295-301
references.pmid: 15371414
references.citation: Biochim Biophys Acta. 2006 Sep-Oct;1757(9-10):1297-300
references.pmid: 16716247
references.citation: J Virol. 2005 Nov;79(21):13735-46
references.pmid: 16227293
references.citation: Mol Biol Cell. 2009 Jan;20(1):306-18
references.pmid: 18971376
references.citation: J Virol. 2000 Nov;74(21):10104-11
references.pmid: 11024139
references.citation: Circ Res. 2006 Aug 18;99(4):415-23
references.pmid: 16857965
references.citation: J Cell Sci. 2006 Dec 15;119(Pt 24):5011-4
references.pmid: 17158907
references.citation: Genes Dev. 2001 Jun 15;15(12):1481-6
references.pmid: 11410528
references.citation: Nature. 2008 Mar 6;452(7183):98-102
references.pmid: 18288109
references.citation: J Virol. 2002 Jan;76(2):802-16
references.pmid: 11752170
references.citation: J Exp Med. 2000 Nov 20;192(10):1391-402
references.pmid: 11085742
references.citation: Cell. 2008 May 16;133(4):681-92
references.pmid: 18485875
references.citation: Nature. 2005 Mar 31;434(7033):652-8
references.pmid: 15800626
references.citation: J Immunol. 2007 Sep 15;179(6):3812-20
references.pmid: 17785818
references.citation: Aging Cell. 2009 Aug;8(4):426-38
references.pmid: 19473119
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 8;106(49):20776-81
references.pmid: 19920172
references.citation: J Biol Chem. 2006 May 19;281(20):14314-20
references.pmid: 16551620
references.citation: Nat Cell Biol. 2007 May;9(5):487-9
references.pmid: 17473857
references.citation: J Biol Chem. 1996 Nov 15;271(46):29073-9
references.pmid: 8910561
references.citation: Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14985-90
references.pmid: 11752447
references.citation: Blood. 2008 Feb 15;111(4):2142-51
references.pmid: 18096765
references.citation: Nat Genet. 2007 Jan;39(1):86-92
references.pmid: 17187068
references.citation: J Exp Med. 1997 Oct 20;186(8):1323-31
references.pmid: 9334372
references.citation: Cell Death Differ. 2008 Mar;15(3):567-79
references.pmid: 18064039
references.citation: Curr Opin Immunol. 2007 Jun;19(3):301-8
references.pmid: 17433871
references.citation: Protein Expr Purif. 1993 Oct;4(5):398-404
references.pmid: 8251751
references.citation: Nature. 1999 Feb 4;397(6718):441-6
references.pmid: 9989411
references.citation: J Biol Chem. 2007 Feb 16;282(7):4545-4552
references.pmid: 17179148
references.citation: Biol Chem. 2006 Jul;387(7):827-37
references.pmid: 16913832
references.citation: Arch Biochem Biophys. 2007 Jun 15;462(2):245-53
references.pmid: 17475204
references.citation: Cell Death Differ. 2000 Dec;7(12):1166-73
references.pmid: 11175253
references.citation: J Biol Chem. 2007 Jun 1;282(22):16223-31
references.pmid: 17374615
references.citation: EMBO J. 2007 Apr 18;26(8):2148-57
references.pmid: 17363894
references.citation: J Exp Med. 2001 Nov 5;194(9):1325-37
references.pmid: 11696597
references.citation: Immunol Cell Biol. 2006 Feb;84(1):72-8
references.pmid: 16405654
references.citation: Biochem Biophys Res Commun. 2008 Jul 4;371(3):391-4
references.pmid: 18439904
references.citation: Mol Cell Biol. 1999 Dec;19(12):8604-15
references.pmid: 10567584
references.citation: Nat Cell Biol. 2007 May;9(5):550-5
references.pmid: 17417626
references.citation: J Mol Biol. 2007 Mar 16;367(1):65-79
references.pmid: 17241641
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W208-13
references.pmid: 15980455
references.citation: Nature. 2004 Jan 29;427(6973):461-5
references.pmid: 14749836
references.citation: Cell. 2007 Nov 2;131(3):476-91
references.pmid: 17981116
references.citation: Microbiol Immunol. 2003;47(1):91-9
references.pmid: 12636258
references.citation: J Biol Chem. 2004 May 21;279(21):22020-9
references.pmid: 15014070
references.citation: Science. 2008 Oct 10;322(5899):268-71
references.pmid: 18845757
references.citation: J Biol Chem. 2005 Apr 22;280(16):16383-92
references.pmid: 15713684
references.citation: J Biol Chem. 2006 Apr 14;281(15):10153-63
references.pmid: 16478725
references.citation: Acta Paediatr Suppl (Upps). 1956 Feb;45(Suppl 105):1-78
references.pmid: 13326376
references.citation: Leukemia. 2004 Oct;18(10):1671-80
references.pmid: 15356645