Granzyme M targets topoisomerase II alpha to trigger cell cycle arrest and caspase-dependent apoptosis

Granzyme M: behind enemy lines

“…31 To determine GrM substrate specificity in a more physiological setting, we have recently used COFRADIC-based N-terminal positional proteomics coupled with a triple L-Arg SILAC (stable isotope labeling with amino acids in cell culture) labeling strategy to determine GrM substrates in living human HeLa tumor cells. 32 Only 25 substrates with a Met or a Leu at the P1 are identified, and the consensus cleavage site in these substrates displays an almost perfect correlation with the previously identified substrate specificities of GrM (Figure 1). Together, these data confirm that hGrM is a highly specific serine protease with a specificity that extends beyond its preferences for P4-P1.…”

“…33 In contrast, several other studies have shown that hGrM treatment can lead to phosphatidylserine exposure, DNA fragmentation, targeting of the mitochondria, and caspase activation. 32,34,[44][45][46] Some classical hallmarks of apoptosis (such as phosphatidylserine exposure, DNA fragmentation, and mitochondrial damage) can be the result of caspase activation, so whether or not caspases are activated in response to hGrM may determine whether or not these apoptotic hallmarks are observed. Differences between studies may be due to the method used to produce recombinant hGrM (in Escherichia coli or Pichia pastoris), the applied hGrM concentrations, or the means used for intracellular delivery of hGrM (purified perforin, Pro-Ject protein transfection reagent, adenovirus, or the pore-forming protein streptolysin O (SLO)).…”

“…Indeed, we have recently identified DNA topology enzyme topoisomerase II alpha (topoIIa) as a highly efficient hGrM substrate. 32 Cleavage of topoIIa by hGrM leads to a dissection of the protein's catalytic domains from the C-terminal regulatory domain, resulting in nuclear exit of the functional domains. Knockdown of topoIIa induces G2/M cell cycle arrest and subsequently leads to apoptosis.…”

“…31 In addition, complementary positional proteomics have been used to determine the consensus cleavage sites of human and mGrM in K562 tumor cell lysates. 31 Finally, complementary positional proteomics have been used on living HeLa tumor cells treated with the pore-forming perforin-analogue SLO and hGrM 32 lymphocyte clones, for instance, contain similar levels of mGrM mRNA, and both display Met-ase activity. 38 …”

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“…The procedures of apoptosis are formed as follows: Receiving apoptotic signals leads to cytomorphological alterations, the interaction between the intermolecular apoptotic proteins, activation of the caspase enzyme that acts upon cytoskeleton proteins, mitochondria, nuclear membrane or chromatin to cleave proteins and substrates, causing the DNA to fragment, releasing the cytoplasm enclosed by the plasma membrane, eventually leading to cell death (10)(11)(12)(13)(14). Currently, it is known that the extrinsic or death receptor pathway, the intrinsic or mitochondrial pathway and the perforin/granzyme pathway are the major apoptotic pathways to activate caspases (12,(15)(16)(17). The apoptotic process is involved in the maintenance of tissue homeostasis, which can be triggered by a variety of stimuli, including cytokines, hormones, toxic insults and viruses.…”