Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms

A TSC22-like motif defines a novel antiapoptotic protein family

The apoptotic programme is evolutionarily conserved between yeast and metazoan organisms. We have previously identified a number of mammalian cDNAs capable of suppressing the deleterious effects of Bax expression in yeast. We herein report that one such suppressor, named Tsc22(86), represents the C-terminal 86 amino acids of the previously characterized leucine zipper (LZ) motif-containing transcriptional regulator Tsc22. Employing a genome-wide two-hybrid screen, functional genomics, and deletion mutagenesis approaches, we conclude that Tsc22(86)-mediated antiapoptosis is independent of the LZ motif and is likely independent of effects on gene transcription. Rather, a 16-residue sequence within the conserved 56-residue TSC22 domain is necessary for antiapoptosis. The presence of a similar sequence was used to predict an antiapoptotic role for two yeast proteins, Sno1p and Fyv10p. Overexpression and knock-out experiments were used to validate this prediction. These findings demonstrate the potential of studying heterologous proteins in yeast to uncover novel biological insights into the regulation of apoptosis

Identification of novel anti-apoptotic sequences by screening for suppressors of the effects of Bax in yeast

Elucidating novel anti-apoptotic regulatory pathways is central to further understanding the molecular basis of several pathologies, including cancer. The Bcl-2 protein family includes central regulators of the apoptotic process in mammalian cells, such as the antiapoptotic Bcl-2 and the pro-apoptotic Bax. Descriptions of apoptotic phenotypes in yeast induced by certain stressful stimuli, including the heterologous expression of Bcl-2 proteins, indicate the presence of an evolutionarily conserved apoptotic programme. We have previously reported the identification of several mammalian cDNAs effective in preventing the lethal effects of heterologous expression of a pro-apoptotic BAX cDNA in yeast (Yang et al., FEMS Yeast Research 2006; 6:751-762). Here we report that one of the Bax suppressors encodes a novel 156 amino acid variant of the human Vps24 protein, Vps24ß, that lacks the N-terminal lipid binding domain of the well characterized 222 residue Vps24 (Vps24a). We demonstrate that the VPS24ß cDNA represents an expressed transcript that is likely produced by alternative splicing of the human VPS24 gene. Vps24a, but not Vps24ß, prevented the temperature and salt sensitive growth defects observed in a yeast mutant lacking a functional VPS24 gene. In contrast, Vps24ß, but not Vps24a, suppressed the inhibitory effects of Bax on yeast growth. Vps24ß protein also suppressed the effects of Bax in mutants lacking other VPS genes suggesting that a functional ESCRT pathway, of which the yeast Vps24p is an essential component, is not required for Vps24ß function. Taken together, we demonstrate that the human VPS24 gene gives rise to two functionally distinct proteins, one of which is involved in the ESCRT pathway and another novel protein that serves an anti-apoptotic role. The apoptotic programme is evolutionarily conserved between yeast and metazoan organisms. The second complete manuscript included herein involves an additional suppressor, named Tsc22(86), that rLa détermination de nouveau cheminements de normalization anti-apoptotique est essentiel à la compréhension de plusieurs pathologies au niveau moléculaire, incluant le cancer et les maladies du coeur. La famille de protéines Bcl-2 inclu des agents de normalization centrales du processus apoptotique présents dans des cellules mammifères, dont l'anti-apoptotique Bcl-2 et le pro-apoptotique Bax. La description des phénotypes apoptotique contenus dans la levure induit par un stimulus aggressif, incluant l'expression hétérologue des protéines Bcl-2, indique la présence d'un programme apoptotique conservé durant l'évolution de ce dernier. Précédemment, nous avons reporté sur l'identification de plusieurs cADNs plutôt efficaces envers la prévention des éffets néfastes concernant l'expression hétérologue d'un BAX cADN pro-apoptotique dans la levure (Yang, Khoury et al., FEMS Yeast Research 2006; 6:751-762). Içi, je reporte le fait qu'un abrogeur Bax est en mesure d'encoder une nouvelle variante d'acide aminé 156 de la protéine humaine Vps24, nommé Vps24ß, qui n'est pas muni du domaine caractéristique d'aggripage de lipides à N-bornes qui se retrouve généralement dans le résidue 222 du Vps24 (Vps24a). Je démontre que le Vps24ß cADN représente une transcription qui est probablement produit par le processus alternatif d'épissement du gène humain Vps24. C'était le Vps24a, et non le Vps24ß, qui a empêché la croissance des défauts sensible au sel et à la température observés dans une affectation de levure qui ne possédait pas un gène Vps24 fonctionnel. Par contre, le Vps24ß, et non le Vps24a, a supprimé les éffets négatives du Bax envers la croissance de la levure. De plus, la protéine Vps24ß a aussi supprimé les éffets du Bax dans des échantillons le levure mutant manquant d'autres gènes VPS, qui suggèrent qu'une voie ESCRT fonctionnelle, ou le Vps24p est essentiel, n'est pas requis pour le Vps24ß. Pris ensembles, n

Human Thyroid Cancer-1 (TC-1) is a vertebrate specific oncogenic protein that protects against copper and pro-apoptotic genes in yeast

The human Thyroid Cancer-1 (hTC-1) protein, also known as C8orf4 was initially identified as a gene that was up-regulated in human thyroid cancer. Here we show that hTC-1 is a peptide that prevents the effects of over-expressing Bax in yeast. Analysis of the 106 residues of hTC-1 in available protein databases revealed direct orthologues in jawed-vertebrates, including mammals, frogs, fish and sharks. No TC-1 orthologue was detected in lower organisms, including yeast. Here we show that TC-1 is a general pro-survival peptide since it prevents the growth- and cell death-inducing effects of copper in yeast. Human TC-1 also prevented the deleterious effects that occur due to the over-expression of a number of key pro-apoptotic peptides, including YCA1, YBH3, NUC1, and AIF1. Even though the protective effects were more pronounced with the over-expression of YBH3 and YCA1, hTC-1 could still protect yeast mutants lacking YBH3 and YCA1 from the effects of copper sulfate. This suggests that the protective effects of TC-1 are not limited to specific pathways or processes. Taken together, our results indicate that hTC-1 is a pro-survival protein that retains its function when heterologously expressed in yeast. Thus yeast is a useful model to characterize the potential roles in cell death and survival of cancer related genes