Identification and functional characterization of the BAG protein family in Arabidopsis thaliana

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.     

The Salmonella Type III Effector SspH2 Specifically Exploits the NLR Co-chaperone Activity of SGT1 to Subvert Immunity

To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL) effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR) model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.     

Disruption of cellular homeostasis induces organelle stress and triggers apoptosis like cell-death pathways in malaria parasite

A regulated protein turnover machinery in the cell is essential for effective cellular homeostasis; any interference with this system induces cellular stress and alters the normal functioning of proteins important for cell survival. In this study, we show that persistent cellular stress and organelle dysfunction because of disruption of cellular homeostasis in human malaria parasite Plasmodium falciparum, leads to apoptosis-like cell death. Quantitative global proteomic analysis of the stressed parasites before onset of cell death, showed upregulation of a number of proteins involved in cellular homeostasis; protein network analyses identified upregulated metabolic pathways that may be associated with stress tolerance and pro-survival mechanism. However, persistent stress on parasites cause structural abnormalities in endoplasmic reticulum and mitochondria, subsequently a cascade of reactions are initiated in parasites including rise in cytosolic calcium levels, loss of mitochondrial membrane potential and activation of VAD-FMK-binding proteases. We further show that activation of VAD-FMK-binding proteases in the parasites leads to degradation of phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), a known target of metacaspases, as well as degradation of other components of spliceosomal complex. Loss of spliceosomal machinery impairs the mRNA splicing, leading to accumulation of unprocessed RNAs in the parasite and thus dysregulate vital cellular functions, which in turn leads to execution of apoptosis-like cell death. Our results establish one of the possible mechanisms of instigation of cell death by organelle stress in Plasmodium.Malaria is a major healthcare problem worldwide resulting in an estimated 0.65 million deaths every year. Present strategy of malaria control is totally dependent on pharmacological treatments and there is a constant need to identify new drug targets involved in important metabolic pathways in the parasite.¹ The cellular machinery responsible for protein quality control and folding is essential for cellular homeostasis and survival of eukaryotic cells. The protein quality control is particularly important for malaria parasites because of its high replication rate, high temperature stress and high load on endoplasmic reticulum (ER) because of large amount of proteins that are to be secreted or exported to the host cytosol. In eukaryotic cells, inhibition of 26 S proteasome is one of the major causes for low clearance of unfolded proteins from ER and therefore leads to ER stress. ER stress response may help the cell to survive through the stress, it can also trigger apoptosis when high levels of unfolded proteins persist for a longer time.² We have earlier shown that disruption of an important metabolic pathway of the parasite can incite the parasite to undergo apoptosis-like cell death.³ A number of other studies have suggested that apoptosis-like cell death can be induced in Plasmodium falciparum by different anti-malarial drugs, antibiotics and other small molecules.^{4, 5} However, the mode of induction of cell death and different cascade of molecular/cellular events leading to apoptosis-like cell death in the parasite are not clearly understood.In this study, we have assessed cellular stress induced by proteasome inhibition on asexual stage P. falciparum parasites. Global quantitative proteomic analyses identified putative pro-survival pathways in the parasites under cellular stress. We further show that persistent proteasome inhibition cause parasite cell death, which is mediated by a cascade of molecular and cellular events. Overall, our results highlight a probable mechanism of cell death and survival in Plasmodium under cellular stress.     

The redox-sensing quinone reductase Lot6p acts as an inducer of yeast apoptosis

Mammalian NAD(P)H:quinone oxidoreductases such as human NQO1 act as inducers of apoptosis. Quinone reductases generated interest over the last decade due to their proposed function in the oxidative stress response. Furthermore, human NQO1 was reported to regulate p53 stability and p53-dependent apoptosis through regulation of cellular oxidation–reduction events. In this study, we have used low concentrations of hydrogen peroxide (0.4 and 0.6 mM) to induce apoptosis-like cell death in wild type, an LOT6 overexpressing and a Δ lot6 yeast strain to monitor cell survival. Using this approach, we demonstrate that yeast quinone reductase Lot6p, an orthologue of mammalian quinone reductases, plays a pivotal role in apoptosis-like cell death in Saccharomyces cerevisiae . Overexpression of LOT6 results in enhanced cell death, as shown by an investigation of the morphological hallmarks of apoptosis-like fragmentation of DNA and externalization of phosphatidylserine, whereas the deletion strain displays a deficiency in apoptosis-like cell death as compared with the wild type. Thus, we propose that Lot6p is directly involved in the control of the apoptosis-like cell death in yeast.     

Apoptosis-like yeast cell death in response to DNA damage and replication defects

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.     

The nucleolar protein SURF-6 is essential for viability in mouse NIH/3T3 cells

SURF-6 is a bona fide nucleolar protein comprising an evolutionary conserved family that extends from human to yeast. The expression of the mammalian SURF-6 has been recently found to be regulated during the cell cycle. In order to determine the importance of SURF-6 in mammalian cells, we applied the Tet-On system to regulate conditionally, in response to tetracycline, the expression of an antisense RNA (asRNA) that targets Surf-6 mRNA in mouse NIH/3T3 cells. Induced Surf-6 asRNA caused an effective depletion of SURF-6 protein resulted in cell death and in an apparent arrest in the G1 phase of the cell cycle. These results provide for the first time evidence that expression of SURF-6 is essential for mammalian cell viability, and suggest that SURF-6 might participate in the progression of cell cycle.     

Yeast cell death caused by mutation of the OST2 gene encoding the epsilon-subunit of Saccharomyces cerevisiae oligosaccharyltransferase

An essential epsilon-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 degrees C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.     

Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae.

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.     

Yeast Cell Death Caused by Mutation of the OST2 Gene Encoding the ε-Subunit of Saccharomyces cerevisiae Oligosaccharyltransferase

An essential ε-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 °C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

Apoptosis-like death in trypanosomatids: search for putative pathways and genes involved

Members of the Trypanosomatidae family comprises species that are causative of important human diseases such as Chagas'disease, Leishmaniasis and sleeping sickness. A wealth of evidence has accumulated that illustrates the ability of these unicellular organisms to undergo, with or without induction (stress conditions), a cell death with some features resembling apoptosis-like phenomenon. However, despite the apparent phenotypic similarities between the apoptosis-like death of kinetoplastids and mammalian nucleated cell programmed cell death (PCD), the pathways seem to differ significantly. This review analyses some of the current data related to the cell death in trypanosomatids. Special attention is given to members of conserved protein families demonstrating remarkable diversity and plasticity of function [i.e. elongation factor-1 subunits α and γ ; and the Silent Information Regulator (SIR2)-related gene, showed to be associated with resistance to apoptosis-like death in Leishmania]. The elucidation of the molecular events which tightly regulated the processes of growth arrest, differentiation and death of Trypanosoma cruzi, Leishmania spp and African trypanosomes, might allow not only to define a more comprehensive view of the cell death machinery in term of evolutionary origin but may also be useful to identify new target molecules for chemotherapeutic drug development and therapeutic intervention.     

The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA

mRNA export is mediated by Mex67p:Mtr2p/NXF1:p15, a conserved heterodimeric export receptor that is thought to bind mRNAs through the RNA binding adaptor protein Yra1p/REF. Recently, mammalian SR (serine/arginine-rich) proteins were shown to act as alternative adaptors for NXF1-dependent mRNA export. Npl3p is an SR-like protein required for mRNA export in S. cerevisiae. Like mammalian SR proteins, Npl3p is serine-phosphorylated by a cytoplasmic kinase. Here we report that this phosphorylation of Npl3p is required for efficient mRNA export. We further show that the mRNA-associated fraction of Npl3p is unphosphorylated, implying a subsequent nuclear dephosphorylation event. We present evidence that the essential, nuclear phosphatase Glc7p promotes dephosphorylation of Npl3p in vivo and that nuclear dephosphorylation of Npl3p is required for mRNA export. Specifically, recruitment of Mex67p to mRNA is Glc7p dependent. We propose a model whereby a cycle of cytoplasmic phosphorylation and nuclear dephosphorylation of shuttling SR adaptor proteins regulates Mex67p:Mtr2p/NXF1:p15-dependent mRNA export.     

Identification and characterization of srp1, a gene of fission yeast encoding a RNA binding domain and a RS domain typical of SR splicing factors.

The SR protein family is involved in constitutive and regulated pre-mRNA splicing and has been found to be evolutionarily conserved in metazoan organisms. In contrast, the genome of the unicellular yeast Saccharomyces cerevisiae does not contain genes encoding typical SR proteins. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD respectively, and a RS (arginine/serine-rich) domain which gave the family its name. We have now cloned from the fission yeast Schizosaccharomyces pombe the gene srp1. This gene is the first yeast gene encoding a protein with typical features of mammalian SR protein family members. The gene is not essential for growth. We show that overexpression of the RNA binding domain inhibits pre-mRNA splicing and that the highly conserved sequence RDAEDA in the RBD is involved. Overexpression of Srp1 containing mutations in the RS domain also inhibits pre-mRNA splicing activity. Furthermore, we show that overexpression of Srp1 and overexpression of the mammalian SR splicing factor ASF/SF2 suppress the pre-mRNA splicing defect of the temperature-sensitive prp4-73 allele. prp4 encodes a protein kinase involved in pre-mRNA splicing. These findings are consistent with the notion that Srp1 plays a role in the splicing process.     

Cell-nonautonomous inhibition of radiation-induced apoptosis by dynein light chain 1 in Caenorhabditis elegans

The evolutionarily conserved process of programmed cell death, apoptosis, is essential for development of multicellular organisms and is also a protective mechanism against cellular damage. We have identified dynein light chain 1 (DLC-1) as a new regulator of germ cell apoptosis in Caenorhabditis elegans. The DLC-1 protein is highly conserved across species and is a part of the dynein motor complex. There is, however, increasing evidence for dynein-independent functions of DLC-1, and our data describe a novel dynein-independent role. In mammalian cells, DLC-1 is important for cellular transport, cell division and regulation of protein activity, and it has been implicated in cancer. In C. elegans, we find that knockdown of dlc-1 by RNA interference (RNAi) induces excessive apoptosis in the germline but not in somatic cells during development. We show that DLC-1 mediates apoptosis through the genes lin-35, egl-1 and ced-13, which are all involved in the response to ionising radiation (IR)-induced apoptosis. In accordance with this, we show that IR cannot further induce apoptosis in dlc-1(RNAi) animals. Furthermore, we find that DLC-1 is functioning cell nonautonomously through the same pathway as kri-1 in response to IR-induced apoptosis and that DLC-1 regulates the levels of KRI-1. Our results strengthen the notion of a highly dynamic communication between somatic cells and germ cells in regulating the apoptotic process.