Human VAMP3 Suppresses or Negatively Regulates Bax Induced Apoptosis in Yeast

Abstract

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).

Keywords: VAMP3 rescue Bax; Yeast and VAMP3; apoptosis; human VAMP3; yeast apoptosis.

Figure 1

The growth assays on the solid and liquid media and death assay (Phloxin B assay). (A,D) Growth of yeast strains in solid agar plates over 72 h, in the glucose-containing minimal medium (S.D.), and galactose containing minimal medium (S.G.). The 4 strains on the upper half of the two plates are Bax(LEU2)VAMP3 (A) and Bax(LEU2)Bcl-xL (D) transformants (1), containing an episomal 2µ-plasmid plasmid that encodes the HA-tagged VAMP3 (A) and Bcl-xL (D) genes and a Bax expression cassette integrated at the LEU2 locus. The 4 strains, in the lower half of the plates (A,D), are Bax(LEU2)–(2) transformants that contain the Bax expression cassette and an empty plasmid. (B,E) Growth of yeast cells, Bax + VAMP3 (B) and Bax + Bcl-xL (E), in the minimal liquid medium containing galactose, throughout 48 h along with the control strain. A two-tailed paired sample t-test shows, statistically, that there was a significant difference (p < 0.05). (C) The percentage of cell death in strains Bax + VAMP3, Bax + Bcl-xL and control Bax(LEU2)–, after growth in galactose for 48 h. The data represent the mean ± S.D. of three independent experiments. Dead cells stained with phloxine B are shown in (F) The images on the left a representative of cells under fluorescent light, showing death cells (red). A two way ANOVA test shows that there was a significant difference (p < 0.05) in cell death between the control strain and each of Bax + VAMP3 and Bax + Bcl-xL strains, but there was no significant difference (p > 0.05) between Bax + VAMP3 and Bax + Bcl-xL.

Figure 2

Live assay (Hoechst 33,342 dye) and measurement of the mitochondrial membrane potential. (A) Visualisation of yeast cells that coexpress Bax and VAMP3 and Bcl-xL or Bax alone, after staining with Hoechst 33,342 dye. The images on the left are representative of cells under fluorescent light, showing live cells (blue). (B) Quantification of the mitochondrial membrane potential of yeast strains, Bax(LEU2)VAMP3, which coexpressed Bax and VAMP3, Bax(LEU2)Bcl xL, which coexpressed Bax and Bcl xL, and Bax(LEU2), which expresses Bax alone, using a fluorescent plate reader. A two way ANOVA test shows that there was a significant difference (p < 0.05) between the control strain and each of Bax + VAMP3 and Bax + Bcl-xL, but there was a significant difference (p < 0.05) between Bax + VAMP3 and Bax + Bcl-xL. The data represent the mean ± S.D. of three independent experiments. RFLU—Relative Fluorescence Unit.

Figure 3

Reactive oxygen species (ROS) measurement, terminal deoxynucleotidyl transferase (TdT)-facilitated dUTP nick end labelling (TUNEL) assay, and Western blotting. (A) Measurement of ROS produced in the yeast strains Bax(LEU2) + VAMP3, Bax(LEU2) + Bcl xL, and Bax(LEU2) –. A two way ANOVA test shows that there was a significant difference (p < 0.05) between the control strain and each of Bax + VAMP3 and Bax + Bcl-xL, but there was no significant difference (p > 0.05) between Bax + VAMP3 and Bax + Bcl-xL. The data represent the mean ± S.D. of three independent experiments. (B) TUNEL assay comparing the nuclear DNA fragmentations in yeast strains Bax(LEU2)VAMP3, Bax(LEU2)Bcl xL, and Bax(LEU2). The cells Bax(LEU2)VAMP3 and Bax(LEU2)Bcl xL have lower nuclear DNA fragmentation compared to Bax(LEU2)–cells. A two way ANOVA test shows that there was a significant difference (p < 0.05) between the control strain and each of Bax + VAMP3 and Bax + Bcl-xL, but there was no significant difference (p > 0.05) between Bax + VAMP3 and Bax + Bcl-xL. The data represent the mean ± S.D. of three independent experiments. (C) Represent the microscopic image of cells after tunnel assay, Bax(LEU2)VAMP3 and Bax(LEU2)Bcl xL. The left images are representative of cells under fluorescent light, showing dead cells (red) (high nuclear fragmentation). (D) Western blot to monitor the presence of Bax in yeast cells that express only Bax and in cells that coexpress Bax together with VAMP3 and, Bcl-xL. Of total cellular proteins, in cell lysates, 10 µg were loaded on to each lane and the blot was probed with an antibody that recognises the c-Myc-tag, Actin, and HA-tag. RFLU—Relative Fluorescence Unit.