Granzyme A activates another way to die

Abstract

Granzyme A (GzmA) is the most abundant serine protease in killer cell cytotoxic granules. GzmA activates a novel programed cell death pathway that begins in the mitochondrion, where cleavage of NDUFS3 in electron transport complex I disrupts mitochondrial metabolism and generates reactive oxygen species (ROS). ROS drives the endoplasmic reticulum-associated SET complex into the nucleus, where it activates single-stranded DNA damage. GzmA also targets other important nuclear proteins for degradation, including histones, the lamins that maintain the nuclear envelope, and several key DNA damage repair proteins (Ku70, PARP-1). Cells that are resistant to the caspases or GzmB by overexpressing bcl-2 family anti-apoptotic proteins or caspase or GzmB protease inhibitors are sensitive to GzmA. By activating multiple cell death pathways, killer cells provide better protection against a variety of intracellular pathogens and tumors. GzmA also has proinflammatory activity; it activates pro-interleukin-1beta and may also have other proinflammatory effects that remain to be elucidated.

Fig. 1. CTLs derived from GzmA or GzmB knockout mouse splenocytes are comparably cytotoxic

Phytohemagglutinin-activated splenocytes from Gzm B^−/− mice, expressing GzmA (left), or from GzmA^−/− mice, expressing GzmB (right), were tested for their ability to kill concancavalin-coated target cells by ⁵¹Cr release assay. Both cell lines are comparable at killing, suggesting that GzmA and GzmB have similar cytolytic potency when released from intact cells.

Fig. 2. Recombinant GzmA is more active than purified native GzmA

(A) Cytotoxicity of recombinant GzmA, purified in bacteria, was compared to that of GzmA purified from human NK cells by our laboratory (JL) or Froelich (CF) and to GzmA purified from lymphokine activated Tcells (LAK). (B) Recombinant GzmA (rGzmA) and NK cell GzmA were compared to GzmB. The recombinant GzmA is active at 250 nM, similar to GzmB, while the purified proteins have much less activity. This could be due to incomplete processing of the native purified protein in cells, although the reason for the apparent discrepancy needs to be investigated. Figure modified from (75), reprinted with permission.

Fig. 3. GzmA disrupts mitochondrial electron transport by cleaving NDUFS3 in electron transport complex I

(A) Diagram of electron transport in mitochondria depicting the 5 inner membrane-associated electron transport complexes. (B) GzmA wreaks havoc on mitochondrial function. GzmA is actively transported into the mitochondrial matrix by an unknown mechanism that likely involves the TOM-TIM twin translocases. Once inside, GzmA disrupts inner membrane-associated ETC complex I by cutting 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 transmembrane potential, and ATP generation. Despite all these changes, the mitochondrial outer membrane remains intact, and pro-apoptotic proteins in the intermembrane space, including cytochrome c (Cyt C), are not released.

Fig. 4. The SET complex is the key to GzmA-mediated DNA damage

The SET complex, which is normally associated with the ER, translocates to the nucleus in response to the superoxide anion generated by GzmA cleavage of NDUFS3 (Fig. 3). GzmA also concentrates in the nucleus where many of its known substrates reside (Table 1). In the nucleus, 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. These are extended by the SET complex exonuclease TREX1. 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. Figure adapted with permission from (15).