Mammalian prion protein suppresses Bax-induced cell death in yeast

Several lines of evidence suggest that PrP(C), the non-infectious form of the prion protein, may function to protect neurons and other cells from stress or toxicity. In this paper, we report on the use of the yeast Saccharomyces cerevisiae as a model system to assay the cytoprotective activity of PrP(C). The mammalian pro-apoptotic protein, Bax, confers a lethal phenotype when expressed in yeast. Since overexpression of PrP(C) has been found to prevent Bax-mediated cell death in cultured human neurons, we explored whether PrP could also suppress Bax-induced cell death in yeast. We utilized a form of mouse PrP containing a modified signal peptide that we had previously shown is efficiently targeted to the secretory pathway in yeast. We found that this PrP potently suppressed the death of yeast cells expressing mammalian Bax under control of a galactose-inducible promoter. In contrast, cytosolic PrP-(23-231) failed to rescue growth of Bax-expressing yeast, indicating that protective activity requires targeting of PrP to the secretory pathway. Deletion of the octapeptide repeat region did not affect the rescuing activity of PrP, but deletion of a charged region encompassing residues 23-31 partially eliminated activity. We also tested several PrP mutants associated with human familial prion diseases and found that only a mutant containing nine extra octapeptide repeats failed to suppress Bax-induced cell death. These findings establish a simple and genetically tractable system for assaying a putative biological activity of PrP(C).     

Identification of a drought-induced rice gene, OsSAP, that suppresses Bax-induced cell death in yeast

We identified rice genes that might be involved in drought stress tolerance by virtue of their anti-apoptotic activity. Potential anti-apoptosis related genes were identified by screening an Oryza sativa cDNA library derived from drought stressed tissues in a yeast functional assay. About 28 O. sativa cDNAs promoted yeast survival following engagement of Bax-induced apoptosis. An O. sativa cDNA encoding R12H780 was a highly conserved putative senescence-associated-protein (OsSAP). OsSAP was both highly and rapidly expressed in response to drought stress. Additionally, OsSAP was found to be localized to the mitochondria. Overall, OsSAP represents a new type of Bax suppressor related gene and endows multiple stress tolerance in yeast.     

A yeast PAF acetylhydrolase ortholog suppresses oxidative death

Phospholipids containing sn-2 polyunsaturated fatty acyl residues are primary targets of oxidizing radicals, producing proapoptotic and membrane perturbing fragmented phospholipids. The only known phospholipases that specifically select these oxidized and/or short-chained phospholipids as substrates are mammalian group VII phospholipases A2s that were purified and cloned as PAF acetylhydrolases. Platelet-activating factor (PAF) is a short-chained phospholipid, and whether these enzymes actually are PAF hydrolases or evolved as oxidized phospholipid phospholipases is unknown. The fission yeast Schizosaccharomyces pombe, which does not form or use PAF as a signaling molecule, contains an open-reading frame potentially homologous to mammalian group VII phospholipase A2s. We cloned this SPBC106.11c locus and expressed it in distantly related Saccharomyces cerevisiae that lack homologous sequences. The S. pombe locus encoded a functional phospholipase A2, now renamed plg7+, that hydrolyzed PAF and a synthetic oxidized phospholipid. Expression of human type II PAF acetylhydrolase or S. pombe Plg7p enhanced the viability of S. cerevisiae subjected to oxidative stress. We conclude that a single-celled organism with an exceedingly spare genome still expresses an unusually discriminating phospholipase A2, and that selective hydrolysis of phospholipid oxidation products is an early, and critical, way to overcome oxidative membrane damage and oxidant-induced cell death.     

Ectopic expression of an Arabidopsis calmodulin-like domain protein kinase-enhanced NADPH oxidase activity and oxidative burst in tomato protoplasts

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.     

The Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) can function as a dominant suppressor of Bax-induced cell death of yeast.

We identified genes based on screening of an Arabidopsis cDNA library for functional suppressors of mouse Bax-induced cell death of yeast cells. Interestingly, the cDNA encoding AtEBP, known as Arabidopsis thaliana ethylene-responsive element binding protein, was isolated numerous times in the functional screen (82% of all suppressors). Full-length AtEBP and its localization to the nucleus were essential for the suppression of Bax-induced cell death. Morphological abnormality of intracellular network that is a hallmark of Bax-induced cell death was attenuated by expression of AtEBP.     

Truffle volatiles inhibit growth and induce an oxidative burst in Arabidopsis thaliana

The function of fungal volatiles in fungal-plant interactions is poorly understood. The aim here was to address this lack of knowledge, focusing on truffles, ectomycorrhizal fungi that are highly appreciated for their aroma. The effect of volatiles released by truffles was tested on Arabidopsis thaliana in a closed chamber bioassay. The volatiles produced by Tuber melanosporum, Tuber indicum and Tuber borchii fruiting bodies inhibited A. thaliana in terms of root length and cotyledon leaf size, and in some cases induced a bleaching of the seedlings, thus indicating toxicity. Ten synthetic volatiles were tested in a similar way. The strongest inhibitory effect was observed with C(8) molecules such as 1-octen-3-ol, an alcohol with a typical 'fungal smell'. Two of these C(8) compounds were further tested to investigate their mechanism of action. 1-Octen-3-ol and trans-2-octenal induced an oxidative burst (hydrogen peroxide, H(2)O(2)) in the A. thaliana leaves as well as a strong increase in the activities of three reactive oxygen species (ROS)-scavenging enzymes. These results demonstrate that fungal volatiles inhibit the development of A. thaliana and modify its oxidative metabolism. Even though limited to laboratory observations, these results indicate the presence of a hitherto unknown function of fungal volatiles as molecules that mediate fungal-plant interactions.     

A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.     

The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity

In plants, reactive oxygen species (ROS) associated with the response to pathogen attack are generated by NADPH oxidases or apoplastic peroxidases. Antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase (FBP1) cDNA in Arabidopsis thaliana was previously shown to diminish the expression of two Arabidopsis peroxidases (peroxidase 33 [PRX33] and PRX34), block the oxidative burst in response to a fungal elicitor, and cause enhanced susceptibility to a broad range of fungal and bacterial pathogens. Here we show that mature leaves of T-DNA insertion lines with diminished expression of PRX33 and PRX34 exhibit reduced ROS and callose deposition in response to microbe-associated molecular patterns (MAMPs), including the synthetic peptides Flg22 and Elf26 corresponding to bacterial flagellin and elongation factor Tu, respectively. PRX33 and PRX34 knockdown lines also exhibited diminished activation of Flg22-activated genes after Flg22 treatment. These MAMP-activated genes were also downregulated in unchallenged leaves of the peroxidase knockdown lines, suggesting that a low level of apoplastic ROS production may be required to preprime basal resistance. Finally, the PRX33 knockdown line is more susceptible to Pseudomonas syringae than wild-type plants. In aggregate, these data demonstrate that the peroxidase-dependent oxidative burst plays an important role in Arabidopsis basal resistance mediated by the recognition of MAMPs.     

Bax-induced cell death in yeast depends on mitochondrial lipid oxidation.

The oxidant function of pro-apoptotic protein Bax was investigated through heterologous expression in yeast. Direct measurements of fatty acid content show that Bax-expression induces oxidation of mitochondrial lipids. This effect is prevented by the coexpression of Bcl-xL. The oxidation actually could be followed on isolated mitochondria as respiration-induced peroxidation of polyunsaturated cis-parinaric acid and on whole cells as the increase in the amount of thiobarbituric acid-reactive products. Treatments that increase the unsaturation ratio of lipids, making them more sensitive to oxidation, increase kinetics of Bax-induced death. Conversely, inhibitors of lipid oxidation and treatments that decrease the unsaturation ratio of fatty acids decrease kinetics of Bax-induced death. Taken together, these results show that Bax-induced mitochondrial lipid oxidation is relevant to Bax-induced cell death. Conversely, lipid oxidation is poorly related to the massive Bax-induced superoxide and hydrogen peroxide accumulation, which occurs at the same time, as chemical or enzymatic scavenging of ROS does not prevent lipid oxidation nor has any effects on kinetics of Bax-induced cell death. Whatever the origin of mitochondrial lipid oxidation, these data show that it represents a major step in the cascade of events leading to Bax-induced cell death. These results are discussed in the light of the role of lipid oxidation both in mammalian apoptosis and in other forms of cell death in other organisms.     

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.     

Overproduction of outer membrane protein suppresses envA-induced hyperpermeability.

A quantitative study on outer membrane components was performed in a number of envelope mutants of Escherichia coli K-12 exhibition different permeability properties for antimicrobial agents. The envA1 allele causing an increased influx for both hydrophobic and hydrophilic drugs was found to be associated with a deficiency in the amount of lipopolysaccharides. The sefA1 envA1 double mutant was found to have a higher outer membrane buoyant density, apparently due to an increase in protein content. This double mutant was still low in lipopolysaccharide content.     

NSm protein of Rift Valley fever virus suppresses virus-induced apoptosis

Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It can cause severe epidemics among ruminants and fever, myalgia, a hemorrhagic syndrome, and/or encephalitis in humans. The RVFV M segment encodes the NSm and 78-kDa proteins and two major envelope proteins, Gn and Gc. The biological functions of the NSm and 78-kDa proteins are unknown; both proteins are dispensable for viral replication in cell cultures. To determine the biological functions of the NSm and 78-kDa proteins, we generated the mutant virus arMP-12-del21/384, carrying a large deletion in the pre-Gn region of the M segment. Neither NSm nor the 78-kDa protein was synthesized in arMP-12-del21/384-infected cells. Although arMP-12-del21/384 and its parental virus, arMP-12, showed similar growth kinetics and viral RNA and protein accumulation in infected cells, arMP-12-del21/384-infected cells induced extensive cell death and produced larger plaques than did arMP-12-infected cells. arMP-12-del21/384 replication triggered apoptosis, including the cleavage of caspase-3, the cleavage of its downstream substrate, poly(ADP-ribose) polymerase, and activation of the initiator caspases, caspase-8 and -9, earlier in infection than arMP-12. NSm expression in arMP-12-del21/384-infected cells suppressed the severity of caspase-3 activation. Further, NSm protein expression inhibited the staurosporine-induced activation of caspase-8 and -9, demonstrating that other viral proteins were dispensable for NSm's function in inhibiting apoptosis. RVFV NSm protein is the first identified Phlebovirus protein that has an antiapoptotic function.     

HCCR-1, a novel oncogene,encodes a mitochondrial outer membrane protein and suppresses the UVC-induced apoptosis

Background  

The Human cervical cancer oncogene (HCCR-1) has been isolated as a human oncoprotein, and has shown strong tumorigenic features. Its potential role in tumorigenesis may result from a negative regulation of the p53 tumor suppressor gene.     

Prion protein suppresses perturbation of cellular copper homeostasis under oxidative conditions

Prion protein (PrP) binds copper and exhibits superoxide dismutase-like activity, while the roles of PrP in copper homeostasis remain controversial. Using Zeeman graphite furnace atomic absorption spectroscopy, we quantified copper levels in immortalized PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilizes copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilizing cellular copper homeostasis under oxidative conditions.     

Identification of Chinese cabbage sentrin as a suppressor of Bax-induced cell death in yeast

Studies into the cell death program termed apoptosis have resulted in new information regarding how cells control and execute their own demise, including insights into the mechanism by which death-preventing factors can inhibit Bax-induced caspase activation. We investigated high temperature stress-induced cell death in Brassica rapa. Using a yeast functional screening from a Brassica rapa cDNA library, the BH5-127 EST clone encoding an apoptotic suppressor peptide was identified. However, a phylogenic tree showed that BH5-127 clusters within a clade containing SUMO-1 (Small Ubiquitin-like Modifier- 1). BH5-127 was confirmed similar to have function to SUMO-1 as Fas suppression. Expression of BH5-127 showed that substantial suppression of cell death survived on SD-galactose-Leu--Ura- medium. The results suggest that BrSE (Brassica rapa Sentrin EST, BH5-127) is one of the important regulatory proteins in programming cell death, especially in the seedling stage of Chinese cabbage.