Revelation of new properties of pro-apoptotic human proteins, Bax and α-synuclein, using baker's yeast

Apoptosis is a form of programmed cell death which is essential for growth of dividing human cells whereas, in contrast, it is deleterious for post-mitotic cells such as neurons. Bax and α-synuclein are two human proteins which play a role in the induction of neuronal apoptosis in neurodegenerative diseases like Alzheimer’s and Parkinson’s. Human Bax and α-synuclein also induce cell death when expressed in baker’s yeast, Saccharomyces cerevisiae. In this thesis, this particular yeast has been used as a model system to study (a) if pro-apoptotic Bax is able to activate an inactive cytochrome P450 (CYP) expressed in the absence of a P450 reductase (CPR), (b) the effects of co-expression of the two pro-apoptotic proteins Bax and α-synuclein, (c) if mitochondrial DNA-deficient petites spontaneously generated by α-synuclein expression in yeast can ultimately undergo cell death, and (d) the cellular consequences of rescue, from Bax-induced cell death in yeast, by novel anti-apoptotic proteins which had originally been identified in a human hippocampal cDNA library. A CPR abstracts electrons from NADPH and transfers to the active site of a CYP to provide a functional CYP. A yeast strain was genetically engineered to delete the endogenous CPR gene. A human CYP expressed in a CPR-null strain is inactive. It was queried if Bax, which induces apoptosis in yeast and human cells by generating reactive oxygen species (ROS), would substitute for the absence of CPR. Since the generation of ROS by Bax stems from an initial release of electrons, is it possible for these released electrons to be captured by an inactive CYP to make it active once again? Surprisingly it was found that Bax does activate three inactive CYP proteins confirming that it can compensate for CPR’s absence within yeast cells. Quite unexpectedly, the human α-synuclein gene had been identified as an inhibitor of pro-apoptotic Bax using a yeast-based screen of a human hippocampal cDNA library. The screen relies on human Bax killing 100% of yeast cells under certain inducible conditions. Numerous plasmids were constructed with different promoters, which allow expression of wild-type and Parkinson’s disease-related mutant α-synuclein genes, from (i) multi-copy 2µ (episomal) plasmids and (ii) integrative plasmids that compel expression of genes from chromosomal sites in varying copy numbers (1 to 3). All α-synuclein-containing plasmids were introduced, through transformation, into a yeast strain which already contained a chromosomally integrated copy of Bax. It is for the first time that it was observed that, depending on gene dosage, only wild-type α-synuclein is anti-apoptotic while mutant α-synuclein is not. The results also indicate that wild-type α-synuclein has a remarkable ability to manifest two contrasting effects depending on its level of expression: (i) normally, it would negate apoptosis but (ii) when overexpressed, it tends to induce apoptosis which is probably what happens in Parkinson’s disease. The human α-synuclein gene when expressed at low levels in yeast spontaneously forms mitochondrial DNA-deficient rho-minus petite cells. It has been published that petites are generally resistant to cell death. Since α-synuclein is known to induce apoptosis both in human neuronal cells and in yeast which contains functional mitochondria, it was queried if petites, formed by low level expression of α-synuclein, would undergo cell death when higher levels of α-synuclein were expressed. Although it has never been seen before, it appears that petites do undergo cell death leading to the conclusion that α-synuclein mediated cell death, at least in yeast, does not require functional mitochondria. Besides human α-synuclein, there were many other novel genes that were initially identified, which rescue Bax’s pro-apoptotic effects, in the yeast-based human hippocampal cDNA library screen. Three specific genes were chosen for further studies to confirm their role in inhibition of apoptosis. The genes code for proteins involved in protein (i) folding, (ii) secretion and (iii) translation. They were sub-cloned downstream of the constitutive PGK1 promoter (expression from which can occur in the presence of both glucose and galactose), on an episomal vector, and were co-expressed with a chromosomally-integrated Bax gene expression cassette driven by the GAL1 promoter (which is galactose-inducible but repressed in the presence of glucose). The anti-apoptotic proteins of the Bcl-2 family are known to negate the pro-apoptotic properties of Bax. However, the results with the three new genes show that (i) proteins need not belong to the Bcl-2 family to overcome Bax’s death-inducing effects, and (ii) novel anti-apoptotic proteins can be identified using a simple yeast assay. The findings presented here seem to corroborate indirect evidence, published in the literature, which hint that these proteins may be anti-apoptotic.