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A role for heat shock protein 27 in CTL-mediated cell death
P J Beresford1, M Jaju, R S Friedman
1The Center for Blood Research, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
CTL exocytosis of granules containing perforin and granzyme proteases induces apoptotic cell death. Either granzyme A or B can act with perforin to trigger apoptosis. Granzyme B activates a ubiquitous apoptotic cascade induced by caspase cleavage, but the granzyme A pathway is largely unknown. Using affinity chromatography with recombinant mutant inactive granzyme A, we previously isolated two granzyme A-binding proteins, PHAP (putative HLA-associated protein) I and II. PHAP II, a substrate of granzyme A, is degraded within minutes of CTL attack. Two additional cytoplasmic proteins of 27 and 53 kDa bind strongly to the mutant granzyme A column, requiring 6 M urea to elute. Sequencing identified these as the monomer and dimer of hsp27, a small heat shock protein up-regulated by stress and cellular activation. Hsp27 coprecipitates with granzyme A from cytoplasmic lysates and is not a substrate of the enzyme. Hsp27 translocates to the detergent-insoluble fraction of target cells and relocalizes from diffuse cytoplasmic staining to long filamentous fibers, especially concentrated in a perinuclear region, within minutes of CTL attack. Hsp27 may participate in morphologic changes during granule-mediated lysis. Low or absent levels of hsp27 expression in T lymphocytes, even after heat shock, may play a role in CTL resistance to granule-mediated lysis.
Insights
Cytotoxic T lymphocytes (CTLs) induce apoptosis via granule exocytosis. Heat shock protein 27 (Hsp27) interacts with granzyme A, potentially influencing CTL-mediated cell death pathways.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Cytotoxic T lymphocytes (CTLs) induce target cell apoptosis through granule exocytosis, releasing perforin and granzyme proteases.
- While granzyme B's apoptotic pathway is well-defined, the granzyme A pathway remains largely uncharacterized.
- Previous studies identified PHAP I and II as granzyme A-binding proteins, with PHAP II being a substrate degraded during CTL attack.
Purpose of the Study:
- To identify and characterize novel granzyme A-binding proteins involved in CTL-mediated cytotoxicity.
- To elucidate the role of identified proteins in the granzyme A apoptotic pathway.
- To investigate the cellular localization and potential function of heat shock protein 27 (Hsp27) during CTL attack.
Main Methods:
- Affinity chromatography using a recombinant mutant inactive granzyme A.
- Protein identification via sequencing.
- Co-precipitation assays and cellular fractionation to determine protein interactions and localization.
Main Results:
- Two additional cytoplasmic proteins, identified as the monomer and dimer of heat shock protein 27 (Hsp27), were found to bind strongly to granzyme A.
- Hsp27 coprecipitates with granzyme A and does not appear to be a substrate.
- Within minutes of CTL attack, Hsp27 translocates to the detergent-insoluble fraction and reorganizes into filamentous structures, particularly in the perinuclear region.
Conclusions:
- Hsp27 is a novel granzyme A-binding protein that interacts with granzyme A during CTL-mediated cytotoxicity.
- Hsp27's dynamic relocalization suggests a role in the morphological changes associated with granule-mediated lysis.
- Low Hsp27 expression in T lymphocytes may contribute to resistance against CTL-mediated apoptosis.