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Apoptosis is a form of programmed cell death which is essential for the 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. 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. Plasmids were constructed with different promoters, which allow expression of wildtype and Parkinson's disease (PD)-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 (1-3). 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 wildtype α-synuclein is anti-apoptotic while mutant α-synuclein is not. The results also indicate that wildtype 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 PD., (© 2020 The Author(s).)

Human α-synuclein expression in baker's yeast reportedly induces mitochondria-dependent apoptosis. Surprisingly, we find that, under de-repressing conditions of the inducible MET25/GAL1 promoters, yeast cells expressing chromosomally-integrated copies of the human α-synuclein gene are not killed, but spontaneously form respiration-deficient rho-minus (ρ - ) petites. Although yeast cells can undergo cell death (apoptosis) from loss of mitochondrial function, they can also survive without functional mitochondria. Such cells are referred to as ρ 0 or ρ - petites. This study reports that minimal expression of human α-synuclein in yeast, from MET25 / GAL1 promoter, gives rise to ρ - petites. Interestingly, the full expression of α-synuclein, from the same promoters, in α-synuclein-triggered ρ - petites and also in ρ 0 petites (produced by treating ρ + cells with the mutagen ethidium bromide) initiates apoptosis. The percentages of petites increase with increasing α-synuclein gene copy-number. ρ - petites expressing α-synuclein from fully-induced MET25 / GAL1 promoters exhibit increased ROS levels, loss of mitochondrial membrane potential, and nuclear DNA fragmentation, with increasing copies of α-synuclein. Our results indicate that, for the first time in yeast, α-synuclein-triggered apoptosis can occur independently of functional mitochondria. The observation that α-synuclein naturally forms petites and that they can undergo apoptosis may have important implications in understanding the pathogenesis of Parkinson's disease.

Eukaryotic elongation factor 1A1 (eEF1A1) is central to translational activity. It is involved in complexes that form signal transduction with protein kinase C, as well as being a signal transducer and activator of transcription 3. eEF1A1 and eEF1A2 are isoforms of the alpha subunit of elongating factor 1 complex. It has been reported that eEF1A1 is expressed in most human tissues but the brain, skeletal muscle and heart. eEF1A1 has been linked to both apoptosis and anti-apoptotic activities. In this study, eEF1A1 was co-expressed with Bax, a proapoptotic protein via heterologous expression of recombinant DNA in yeast cells. Assays were carried out to monitor the fate and state of yeast cells when eEF1A1 was co-expressed with Bax. The yeast strain (bearing an integrated copy of the Bax gene) was transformed with an episomal 2-micron plasmid that encodes HA-tagged eEF1A1 gene. The resultant strain would allow co-expression of Bax and eEF1A1 in yeast cells, Bax being under the control of the GAL1 promoter, while the PGK1 promoter drives eEF1A1 expression. Bcl 2A1, a known anti-apoptotic protein, was also co-expressed with Bax in yeast cells as a positive control, to study the anti-apoptotic characteristic of eEF-1A1. The part eEF1A1 plays in apoptosis has been contentious, amidst the pro and anti-apoptotic properties of eEF1A1, it was shown clearly, in this study that eEF1A1 portrays only anti-apoptotic property in the presence of pro-apoptotic protein, Bax.

Aggregation/misfolding of α-synuclein and βA4 proteins cause neuronal cell death (NCD) associated with Parkinson's and Alzheimer's disease. It has been suggested that a heat shock protein-90 (Hsp90) inhibitor can prevent NCD by activating the heat shock transcription factor-1 which, in turn, upregulates molecular chaperones such as Hsp70 that targets aggregated/misfolded proteins for refolding/degradation. We have isolated radicicol, an Hsp90 inhibitor, from a fungus occurring in the crevices of marble rocks of Central India. Radicicol, which was found to be a strong antioxidant, was tested for its ability to rescue yeast cells from death induced by expression of wild-type α-synuclein, its more toxic A53T mutant, and βA4. It effectively overcomes wild-type/mutant α-synuclein mediated yeast cell death, concomitantly diminishes ROS levels, reverses mitochondrial dysfunction and prevents nuclear DNA-fragmentation, a hallmark of apoptosis. Surprisingly however, radicicol is unable to rescue yeast cells from death triggered by expression of secreted βA4. Moreover, although radicicol acts as an antioxidant it fails to prevent yeast cell death inflicted by the proapoptotic protein, Bax. Our results indicate that radicicol specifically targets aggregated/misfolded α-synuclein's toxicity and opens up the possibility of using multiple yeast assays to screen natural product libraries for compounds that would unambiguously target α-synuclein aggregation/misfolding., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aggregated Aβ peptides which cause amyloid deposits, a characteristic of Alzheimer's disease (AD), activate a stress response in the endoplasmic reticulum (ER), known as the unfolded protein response, UPR ER . Nascent UPR ER induction helps in reducing ER stress by eliminating accumulated misfolded/aggregated secretory proteins. However, prolonged UPR ER induction may trigger apoptosis. Here we show that, when expressed in yeast with an NH 2 -terminal secretory signal sequence (ss), the 42-amino acid human Aβ42 (h_Aβ42), but not the mouse/ratAβ42 (m_Aβ42) which reportedly does not misfold/aggregate, induces UPR ER as monitored via an eGFP reporter. We also show that expression of ss-h_Aβ42, not ss-m_Aβ42, blocks yeast cell growth, with cells expressing ss-h_Aβ42 manifesting distinctive features of apoptosis such as loss of mitochondrial membrane potential, increase in ROS levels and DNA fragmentation. Screening for suppressors of ss-h_Aβ42-activated UPR ER -eGFP induction, in a computationally-designed 29-compound methoxy-stilbene library, revealed three compounds that reduce >95% of UPR ER -eGFP induction at 5 μM concentration, with EC 50 values of 40-50 nM. Surprisingly, the compounds also rescue yeast cells from ss-h_Aβ42-mediated apoptosis, with EC 50 -s of 50-60 nM. These results provide direct evidence, probably for the first time, that there is a direct correlation between deactivation of UPR ER and attenuation of apoptosis., (Copyright © 2018. Published by Elsevier B.V.)

Objectives This study aimed to investigate the anticancer potential of indigocarpan ( 1), a pterocarpan isolated from Indigofera aspalathoides, a plant found in India which has been used in Ayurveda for centuries for the treatment of oedematous tumours. Methods The antiproliferative activity in a panel of four human cancer cell lines was studied. The mechanism of its antiproliferative activity in human colorectal adenocarcinoma LS174T cells was investigated in detail. Key findings Indigocarpan ( 1) showed antiproliferative activity in a panel of four human cancer cell lines with IC50s ranging from 180 to 250 μ m. Indigocarpan induces p53-dependent p21 upregulation and apoptosis in LS174T cells, upregulates p53 and p21WAF1 protein levels, enhances cleavage of caspase-3 and downregulates cyclin D1, cyclin B1 and PCNA protein levels, indicating its role in modulating cell cycle progression. Indigocarpan also exhibited a strong antioxidative effect in LS174T cells. Conclusions Along with the antiproliferative capacity, the strong antioxidative property of the compound makes it a promising candidate for further development as an anticancer and chemopreventive compound. [ABSTRACT FROM AUTHOR]

Examines the bax-induced release of cytochrome c from yeast mitochondria. Involvement of permeability transition of the inner mitochondrial membrane; Alteration of permeability of the outer mitochondrial membrane to macromolecules; Analysis of the outer-membrane voltage dependent anion channel; Putative component of the permeability transition pore.

We have reported earlier that cytosolic expression of the full-length human apoptosis inducer Bax-α (Bax) in the yeast Saccharomyces cerevisiae suppresses growth and induces mortality in cells containing functional mitochondria. Human Bcl-x(L) overcomes this toxicity. Here we describe that a mutant Bax protein, with a missing membrane anchor region (BaxΔ), also inhibits growth and causes cell death in yeast. However, the death inhibitory proteins Bcl-x(L) and Bcl-2 fail to rescue BaxΔ-mediated growth inhibition under conditions promoting respiration, although they bind BaxΔ in the cell. Results in Jurkat T-cells corroborate that Bcl-x(L) is much less efficient at rescuing mammalian cells from the effect of BaxΔ than from full length Bax. We have also inquired if the respiration-dependent toxicity of Bax and BaxΔ in yeast is nullified by Bcl-x(L)Δ and Bcl-2Δ, molecules which lack membrane anchors but bind Bax in the yeast two-hybrid system. It appears that, under conditions which facilitate respiration in yeast, Bcl-x(L)Δ and Bcl-2Δ are incapable of rescuing both Bax-containing and BaxΔ-containing cells. Our results open up the interesting possibility that there might exist proteins, unrelated to the Bcl-2 family, which could negate death induced by a membrane anchor-free form of Bax. [ABSTRACT FROM AUTHOR]

Apoptosis is an essential process that is regulated genetically and could lead to a serious disease condition if not well controlled. Bax is one of the main proapoptotic proteins and actively involved in programmed cell death. It has been suggested that Bax induced apoptosis in yeast could be obstructed by enhancing vesicular membrane trafficking. Plasma membrane proteins and lipid oxidation were reduced by a vesicle-associated membrane protein (VAMP) when expressed in yeast, suggesting its potential role in repairing membranes. Membrane integrity is crucial, as the loss of membrane integrity will result in the leakage of ions from mitochondria, and ultimately cell death due to overproduction of reactive oxygen species (ROS). Expression of Arabidopsis' VAMP has been linked to antiapoptosis activity. Since plant VAMP has been associated with antiapoptotic activities, this study investigates the possible participation of human VAMP3 in blocking human Bax mediated apoptosis. Some novel genes were identified to rescue Bax's proapoptotic effects, in a yeast-based human hippocampal cDNA library screen. VAMP3 (a gene code for proteins involved in protein secretion) gene was chosen for further study to confirm its role in inhibiting apoptosis. VAMP3 was coexpressed with a chromosomally integrated Bax gene expression cassette driven by the GAL1 promoter. The antiapoptotic proteins of the Bcl-2 family (Bcl xL) were known to negate the proapoptotic properties of Bax. However, the new gene (VAMP3) results show that novel antiapoptotic proteins can be identified using a yeast-based assay. The findings presented here show that human VAMP3 protein has antiapoptotic property and could abrogate Bax induced apoptosis (cell death). [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Herein, we have identified yeast Sec22p (ySec22p), a SNARE protein essential for endoplasmic reticulum to Golgi trafficking, as a suppressor of Bax-induced yeast apoptosis and corroborated published observations that ySec22p suppresses α-synuclein's toxicity in yeast. It has been suggested that compounds which enhance expression, in neurons, of human homologues of ySec22p (Sec22Bp/Sec22p/Sec22A) would prevent synucleinopathies, such as Parkinson's disease. With the aim of finding a small molecule that would mimic ySec22p, a library of natural products consisting of 394-compounds was screened using yeast cells that express either human α-synuclein or human Bax. The antioxidant aegeline, an alkaloid-amide occurring in the leaves of the plant Aegle marmelos Correa, was the only molecule that overcame apoptosis induced by both α-synuclein and Bax in yeast. Besides, aegeline also prevented growth block in cells expressing the more toxic A53T α-synuclein mutant. Restoration of cell growth occurred through inhibition of increased ROS levels, mitochondrial membrane potential loss and nuclear DNA fragmentation, characteristics of apoptosis manifested in α-synuclein or Bax-expressing cells. These results highlight the importance of yeast systems to identify rapidly molecules that may prevent the onset of apoptosis that occurs in Parkinson's disease. [ABSTRACT FROM AUTHOR]