Combining clonogenic survival tests and the measurement of characteristic cell death markers, we show that the proteolytically inactive N-terminal propeptide of the yeast CatD ortholog (Pep4p) suppresses necrosis in a genetically regulated manner, arguing for the involvement of a cell death type known as ‘programmed necrosis'.10, 11, 33, 34
In this case, necrosis does not mainly result from strong cellular injury or physical disruption of the plasma membrane as an accidental, uncontrolled form of cell death (‘classical necrosis'), but rather follows an orchestrated program, similar to the regulated apoptotic self-execution.10, 11
The anti-necrotic effect of Pep4p seems to involve the regulation of polyamine synthesis (), as it goes along with an increase in the intracellular levels of putrescine, spermidine and its biosynthetic precursor SAM. In addition, Pep4p loses its anti-necrotic activity when spermidine biosynthesis is blocked. Interestingly, external administration of spermidine inhibits necrosis during yeast chronological aging in a process involving histone hypoacetylation, a further correlate of the Pep4p anti-necrotic activity.20
Figure 6 Yeast CatD's functions in cell metabolism. Besides its central role in the vacuolar proteolytic system, where it is a pivotal player for general protein turnover and maturation of several vacuolar hydrolases, the yeast ortholog of CatD, Pep4p, also holds (more ...)
The pro-survival effects of spermidine have been linked to autophagy,20
which is tightly connected to lifespan extension29
and Pep4p has a central role in the autophagic process.28
However, our data reveal that autophagy is not required for the anti-necrotic effect of Pep4p, at least during the early phase of chronological aging. Of note, it has been recently reported that Pep4p overexpression confers autophagy-independent cytoprotection upon acetic acid-induced cell death.35
Though the anti-necrotic function of spermidine is largely dependent on autophagy, during early
aging, autophagy-independent backup-mechanisms have been postulated to exist.20
Given the autophagy-independent yet polyamine-associated nature of the anti-necrotic effect herein described, it is probable that these backup-mechanisms might be related, at least in part, to Pep4p-driven inhibition of programmed necrosis.
Intriguingly, both CatD hyperexpression, as well as increased production of polyamines are observed in many human cancers.4, 36
The propeptide of CatD has been specifically associated to enhanced proliferation and survival of tumor cells3, 5
and polyamines have been shown to mediate tumor promotion and progression.36
It is tempting to speculate that, like Pep4p, human CatD might represent a positive regulator of polyamine biosynthesis and thus a unique target for therapeutic intervention on cellular or tissue polyamine pools. In addition, this study opens doors to investigate CatD orthologues in pathological unicellular organisms as a target for therapeutic necrosis induction.
Our results further provide evidence that Pep4p exerts dual cytoprotective activities involving an anti-necrotic and an anti-apoptotic function. Deletion of PEP4
causes premature death during chronological aging resulting from a combination of necrotic and apoptotic death. Overexpression of proteolytically inactive Pep4p in the PEP4
disruptant specifically reduces the necrotic population and significantly retards the onset of cell death. However, substantial premature demise is not prevented and the apoptotic population remains unchanged. Thus, the requirement of Pep4p for healthy aging is predominantly mediated by its proteolytic function (), which exhibits anti-apoptotic characteristics. However, this anti-apoptotic activity is not involved in lifespan extension; wild type and enzymatically inactive Pep4p (or the sole Pep4p propeptide) confer a similar degree of improved survival through necrosis inhibition (but not apoptosis inhibition) when they are overexpressed in cells that contain baseline levels of endogenous, catalytically active Pep4p. Of note, CatD-deficient neurons exhibit, besides autophagic stress (aberrant accumulation of autophagosomes), also apoptotic markers.37, 38
However, apoptosis does not seem to be critical for neurodegeneration, suggesting alternative death mechanisms involved in neuronal death following CatD-deficiency.39
It would be interesting to analyze if a putative increase in programmed necrosis is responsible for neuron demise upon CatD deficiency.
Under the conditions tested here, Pep4p overexpression protects cells from hydrogen peroxide-induced cell death, which is in line with the enhanced resistance to oxidative stress described for long-lived mutants. Nevertheless, it is conceivable that, like its human counterpart, also yeast CatD might bear a double function in regard to PCD. Thus, it remains to be explored to which extent Pep4p may have a regulatory role as an apoptotic and/or necrotic executioner (in addition to its pro-survival roles) (). In that case, context-dependent regulatory processes could determine whether the pro-survival or the lethal function of Pep4p prevails.
In conclusion, this study uncovers a novel pro-survival function of yeast CatD that is associated with polyamine regulation and specifically exhibits anti-necrotic characteristics, establishing that the capacity of yeast cells to undergo programmed necrosis is under tight molecular control.