Proteolytic regulation of PGAM5 in non-apoptotic cell death

The understanding of cell death has shifted from a distinction between apoptosis and necrosis to a diverse landscape of regulated pathways. This thesis focuses on two such pathways, necroptosis and parthanatos, both relevant in conditions like hypoxia-induced damage and neurodegenerative disease.

Necroptosis, described in 2005, depends on RIPK3 and MLKL, but other factors also modulate this process. Among them, PGAM5 was initially proposed as essential, though later studies challenged this. Here, PGAM5 was shown to accumulate strongly in murine L929Ts C47 fibrosarcoma cells undergoing necroptosis. This applied to both endogenous and transfected PGAM5, suggesting proteolytic regulation rather than transcriptional regulation. Consistent with this, inhibition of the proteasome - via MG-132, the E1 ubiquitin-activating enzyme inhibitor Pyr-41, or siRNA targeting PSMC4 - led to marked increases in PGAM5 levels. These findings support a proteasome-dependent regulation of PGAM5. Although the precise role of elevated PGAM5 during necroptosis remains unclear.

A second aim was to examine whether the mitochondrial rhomboid protease PARL contributes to necroptosis. PARL typically cleaves PINK1 but can shift to PGAM5 when mitochondrial membrane potential is lost. Mouse embryonic fibroblasts lacking PARL or reconstituted with PARL-FLAG showed no difference in necroptotic sensitivity, suggesting PARL is dispensable for this pathway.

Parthanatos, driven by PARP1 activity, was also examined to determine whether PGAM5 or the mitochondrial protease HtrA2 participate in this death mechanism. L929Ts cells deficient in PGAM5 or HtrA2 were exposed to the alkylating agent MNNG. While PGAM5 loss had no effect, HtrA2 deficiency strongly reduced parthanatos, indicating that HtrA2 is involved in this cell death pathway and represents a promising target for further study.

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