Granzyme Pathway
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Granzyme Pathway

Granzymes are a family of conserved serine proteases stored within the cytotoxic granules of cytotoxic lymphocytes (CLs) whose functions were once believed to be primarily involved in immune-targeted cell death. There are 5 granzymes expressed in humans: granzymes A, B, H, K, and M. Granzymes A and B are the most abundant granzymes (Ref.1). Release of the contents of cytotoxic granules into the immunological synapse formed between the killer cell and its target cell is important for immune elimination of viruses, intracellular bacteria, and tumors (Ref.3). GzmA activates caspase-independent death morphologically identical to apoptosis, characterized by single-stranded DNA damage, mitochondrial dysfunction, and loss of cell membrane integrity whereas; GzmB activates apoptosis by cleaving caspases and some key caspase pathway substrates (Ref.3). Although GzmA and GzmB follow distinct pathways to trigger cellular death, they share a common mechanism to get into the target cell. The cytotoxic granules of CTLs and NK cells are specialized secretory lysosomes contain perforin and granzymes (Ref.1). Upon exocytosis of granule contents toward the immune synapse, perforin forms small pores in the target cell membrane, allowing the passage of Ca2+ but not larger molecules such as granzymes. The Ca2+ influx triggers the damaged membrane response, resulting in endocytic uptake of CTL/NK cell membranes and associated granule contents. The newly formed early endosomes fuse to form large vesicles termed gigantosomes containing perforin and Gzms. Once released in to cytoplasm GzmA and GzmB follow their respective path (Ref.4).

Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis but has unique substrates and mediators (Ref.2).After entering the cytosol of target cells, GzmA is specifically imported into mitochondria through the Tim/Tom/Pam import pathway. Once inside, GzmA disrupts inner membrane-associated ETC complex I by cutting NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), which is located in the neck of the stalk of complex I that protrudes into the matrix. Disrupting complex I leads to ROS production and interferes with electron transport, the maintenance of the mitochondrial trans-membrane potential, and ATP generation. The SET complex contains three nucleases [the base excision repair (BER) endonuclease Ape1, an endonuclease NM23-H1 and a 5'-3' exonuclease Trex1, the chromatin modifying proteins SET and pp32, which are also inhibitors of PP2A, and a DNA binding protein called HMGB2, that recognizes distorted DNA. GzmA cleaves 3 components of the SET complex: SET, HMGB2, and APE1. SET is an inhibitor of the SET complex endonuclease NM23-H1. SET cleavage activates NM23-H1 to make single-stranded DNA nicks. GzmA also degrades the linker histone H1 and removes the tails from the core histones, which opens up chromatin and makes it accessible to these nucleases (Ref.5,2 and 6).

Once in the cytoplasm, GzmB targets multiple protein substrates, resulting eventually into apoptotic demise of the target cell both in caspase dependant and independent manner. One of the major substrate of GzmB is Pro-caspase-3. GzmB-activated caspase-3results in the processing of several cellular substrates integral to eliciting the apoptotic phenotype. Moreover, the apoptotic pro-caspases including procaspase-6, 7, 8, 10, 9,and 2 are also serve as substrates for the active GzmB (Ref.7).The mitochondrial pathway by which GzmB induce cell death is through the cleavage of the BH3-only protein Bid into a truncated form, tBid, which then translocates to the mitochondrion and disrupts mitochondrial membrane integrity through interactions with the pro-apoptotic proteins BAX and/ or BAK. In this instance, the pro-apoptotic proteins BAX and/ or BAKmediate mitochondrial outer membrane permeabilization (MOMP) and release several pro-apoptotic intermembrane mitochondrial proteins such as cyt-c, second mitochondria-derived activator of caspases (Smac), high temperature requirement A2 (HtrA2)/ Omi serine protease, apoptosis inducing factor (AIF) and endonuclease-G (Endo-G) (Ref.7,8 and1). Cytochrome c release stimulates the formation of a macromolecular complex consisting of cytochrome c, dATP, apaf-1, and pro-caspase-9known as the apoptosome, which results in the activation of caspase-9and the subsequent activation of caspases-3,6,7culminating in proteolysis of multiple proteins. GzmB also cleaves the anti-apoptotic protein Mcl-1, which results in the release of the pro-apoptotic Bcl-2family member Bim, followed by mitochondrial outer membrane permeabilization and cytochrome c release (Ref.8). ENDOG, released by the action of GzmB-cleaved BID, can induce oligonucleosomal DNA damage (Ref.1).In addition to BID -mediated mitochondrial damage GzmB also directly disrupts the mitochondrial transmembrane potential in a caspase and BID independent manner (Ref.1). Within the nucleus, GzmB directly cleaves the subunit A of DNA fragmentation factor (DFF), which is a hetero-dimer, consisting of the inhibitor/chaperone subunit A (DFFA / ICAD) and the nuclease subunit B (DFFB / CAD), and is pre-bound to DNA. After cleavage, DFFB homodimerizes and cleaves both strands of the genomic DNA. The GzmB-mediated DFF activation can be an alternative way leading to apoptotic DNA fragmentation in cancer cells which are unable to translocate the active caspase-3, a main DFF activator, into the nucleus or carry a loss-of-function mutation of the CASP3 gene (Ref.7).

Though GzmA activates caspase-independent programmed cell death that morphologically resembles apoptosis but has unique substrates and mediators and GzmB activates caspase cell death pathways by initiating effector caspase cleavage and by directly cleaving some key caspase pathway substrates, such as bid and DFFA, few substrates, such as PARP-1, which recruits DNA repair factors and lamin B, that maintain the nuclear envelope, are common to both GzmA and GzmB (Ref.2).