Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells use perforin to deliver serine proteases called granzymes into the cytosol of virally infected or tumor target cells to induce apoptosis. Mice deficient in perforin are severely immunocompromised as are humans with familial hemophagocytic lymphohistiocytosis due to mutations in the perforin gene. The mechanism of perforin action is poorly understood and is the goal of this proposal. Perforin multimerizes in membranes in a calcium-dependent manner. The original model of perforin action by forming pores in the plasma membrane has been called into question by experiments that show that perforin-treated cells are impermeant to low molecular weight dyes and are too small to allow granzymes through. In preliminary data perforin was found to induce rapid endocytosis of granzymes to form large vesicles (""""""""gigantosomes"""""""") that lyse to release granzymes into the target cell cytosol. This proposal will test a model of perforin acting via triggering endocytosis and then endosomolysis. It will identify the cellular trafficking pathways involved in perforin delivery of granzymes and determine the biochemical requirements for perforin activity. Mutational studies will identify the critical perforin domains required for membrane insertion, multimerization and endosomolysis. Structural studies will focus on the perforin C2 domain, hypothesized to undergo a structural change in response to binding Ca++, which is likely to be the first step in membrane binding and insertion. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063430-02
Application #
7184366
Study Section
Cellular and Molecular Immunology - B (CMI)
Program Officer
Miller, Lara R
Project Start
2006-02-15
Project End
2011-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
2
Fiscal Year
2007
Total Cost
$645,316
Indirect Cost
Name
Immune Disease Institute, Inc.
Department
Type
DUNS #
059709394
City
Boston
State
MA
Country
United States
Zip Code
02115
Thiery, Jerome; Keefe, Dennis; Boulant, Steeve et al. (2011) Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells. Nat Immunol 12:770-7
Lieberman, Judy (2010) Granzyme A activates another way to die. Immunol Rev 235:93-104
Thiery, Jerome; Keefe, Dennis; Saffarian, Saviz et al. (2010) Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis. Blood 115:1582-93
Thiery, Jerome; Walch, Michael; Jensen, Danielle K et al. (2010) Isolation of cytotoxic T cell and NK granules and purification of their effector proteins. Curr Protoc Cell Biol Chapter 3:Unit3.37
Sharma, Alok K; Ye, Liwen; Zolotarev, Alexander S et al. (2009) NMR assignment and secondary structure of the STAS domain of Rv1739c, a putative sulfate transporter of Mycobacterium tuberculosis. Biomol NMR Assign 3:99-102
Chowdhury, Dipanjan; Lieberman, Judy (2008) Death by a thousand cuts: granzyme pathways of programmed cell death. Annu Rev Immunol 26:389-420
Nieland, Thomas J F; Shaw, Jared T; Jaipuri, Firoz A et al. (2008) Identification of the molecular target of small molecule inhibitors of HDL receptor SR-BI activity. Biochemistry 47:460-72