| Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells defend against viruses and transformed cells through the release of pro-apoptotic proteins from cytotoxic granules into the immune synapse formed with the target cell to trigger apoptosis. Granzyme A (GzmA), the most abundant protease in cytotoxic granules, induces caspase-independent cell death. GzmA generates reactive oxygen species from mitochondria by cleaving a component of electron transport complex I, then concentrates in the nucleus to cause single-stranded DNA damage by activating two nucleases (NM23-H1 and TREX1). GzmA destroys nuclear envelope integrity and chromosomal stability through the targeted cleavage of lamins and histones.;To understand why GzmA concentrates in the nucleus, we analyzed the nuclear proteome of nuclei treated with GzmA to identify and validate novel nuclear substrates of GzmA. GzmA cleaves many RNA binding proteins, including multiple heterogeneous nuclear ribonucleoproteins (hnRNPs). GzmA cleaves hnRNP A1 after Arg196, Arg232, and Arg284, separating the main RNA binding domains from its nuclear localization domain: GzmA-generated hnRNP A1 cleavage products and endogenous hnRNP A1 mislocalize to the cytoplasm during GzmA attack. This mislocalization is dependent upon GzmA-cleavage of hnRNP A1 since an uncleavable mutant of hnRNP A1 does not mislocalize in response to GzmA. Because hnRNPs and other predicted GzmA substrates are involved in many steps of mRNA maturation and export, we hypothesized that mRNA processing or export would be affected by GzmA. In fact, newly synthesized RNA of cells treated with GzmA plus the delivery molecule, perforin, was retained in the nucleus, indicating that GzmA may disable mRNA export.;Many of these hnRNPs are also degraded in caspase-dependent cell death. Using extrinsic and intrinsic activators of caspases, we show that multiple hnRNPs are degraded in response to caspase activation. Additionally, hnRNP A1 mislocalizes to the cytoplasm during caspase-mediated cell death, indicating that caspases may be similarly targeting hnRNPs to affect regulation of RNA.;This dissertation shows that multiple hnRNPs are targets of both caspase-dependent and -independent cell death pathways. The convergence of these two pathways targeting hnRNP proteins suggests that altering mRNA processing and trafficking may be a common feature of programmed cell death. |
