The Cellular Concentration of the Yeast Ure2p Prion Protein Affects Its Propagation as a Prion

The [URE3] yeast prion is a self-propagating inactive form of the Ure2p protein. We show here that Ure2p from the species Saccharomyces paradoxus (Ure2p_Sp) can be efficiently converted into a prion form and propagate [URE3] when expressed in Saccharomyces cerevisiae at physiological level. We found however that Ure2p_Sp overexpression prevents efficient prion propagation. We have compared the aggregation rate and propagon numbers of Ure2p_Sp and of S. cerevisiae Ure2p (Ure2p_Sc) in [URE3] cells both at different expression levels. Overexpression of both Ure2p orthologues accelerates formation of large aggregates but Ure2p_Sp aggregates faster than Ure2p_Sc. Although the yeast cells that contain these large Ure2p aggregates do not transmit [URE3] to daughter cells, the corresponding crude extract retains the ability to induce [URE3] in wild-type [ure3-0] cells. At low expression level, propagon numbers are higher with Ure2p_Sc than with Ure2p_Sp. Overexpression of Ure2p decreases the number of [URE3] propagons with Ure2p_Sc. Together, our results demonstrate that the concentration of a prion protein is a key factor for prion propagation. We propose a model to explain how prion protein overexpression can produce a detrimental effect on prion propagation and why Ure2p_Sp might be more sensitive to such effects than Ure2p_Sc.     

Prion Domain of Yeast Ure2 Protein Adopts a Completely Disordered Structure: A Solid-Support EPR Study

Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer’s, Parkinson’s, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer’s amyloid-β monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.     

A Specific Population of Abnormal Prion Protein Aggregates Is Preferentially Taken Up by Cells and Disaggregated in a Strain-Dependent Manner

Prion diseases are characterized by the conversion of the soluble protease-sensitive host-encoded prion protein (PrP^C) into its aggregated, protease-resistant, and infectious isoform (PrP^Sc). One of the earliest events occurring in cells following exposure to an exogenous source of prions is the cellular uptake of PrP^Sc. It is unclear how the biochemical properties of PrP^Sc influence its uptake, although aggregate size is thought to be important. Here we show that for two different strains of mouse prions, one that infects cells (22L) and one that does not (87V), a fraction of PrP^Sc associated with distinct sedimentation properties is preferentially taken up by the cells. However, while the fraction of PrP^Sc and the kinetics of uptake were similar for both strains, PrP^Sc derived from the 87V strain was disaggregated more rapidly than that derived from 22L. The increased rate of PrP^Sc disaggregation did not correlate with either the conformational or aggregate stability of 87V PrP^Sc, both of which were greater than those of 22L PrP^Sc. Our data suggest that the kinetics of disaggregation of PrP^Sc following cellular uptake is independent of PrP^Sc stability but may be dependent upon some component of the PrP^Sc aggregate other than PrP. Rapid disaggregation of 87V PrP^Sc by the cell may contribute, at least in part, to the inability of 87V to infect cells in vitro.     

Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils

Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed positive cooperativity for the substrate glutathione in both the soluble native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ion and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the molecular structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.The tripeptide glutathione (GSH)² is abundant in the cell. It plays an important role as a reducing agent in vivo, such as in endogenous free radical scavenging, reversible protein S-glutathionylation, and the reduction of the active sites of enzymes. One major class of enzyme that uses GSH as a reductant is glutaredoxin (GRX), which is a small protein involved in reduction of ribonucleotide reductase for the formation of deoxyribonucleotides for DNA synthesis (1), reduction of 3′-phosphoadenylylsulfate reductase (2) for generation of sulfite, signal transduction, and protection against oxidative stress (3). GRXs are ubiquitous thiol-disulfide oxidoreductases that belong to the thioredoxin superfamily (4). GRXs also show dehydroascorbic acid (DHA) reductase (DHAR) activity (5). Yeast Saccharomyces cerevisiae has at least seven GRXs, which can be divided into two classes according to the number of cysteines in their active site motif: dithiol GRXs with the active site motif CXXC and monothiol GRXs with the motif CXXS (6–9). The dithiol GRXs catalyze protein disulfide reduction using a dithiol mechanism for which both the active site cysteines are essential. On the other hand, both the dithiol and monothiol GRXs can catalyze the reduction of GSH-protein mixed disulfides using a monothiol mechanism that only requires the N-terminal active site cysteine. This reaction and mechanism is important for reversible protein glutathionylation in redox signaling and oxidative stress (10).Glutathione S-transferases (GSTs) are a large versatile family of enzymes with multiple functions, particularly associated with cellular detoxification (11). In terms of overall structure, they belong to the thioredoxin superfamily, like GRX (4). In general, GSTs catalyze the conjugation of reduced GSH to hydrophobic substrates containing an electrophilic atom. In addition, GSTs bind a broad spectrum of ligands and show many other functions. For example, some GSTs show overlapping functions with glutathione-dependent peroxidases (GPxs), which use GSH to reduce hydrogen peroxide and/or organic hydroperoxides and thus are responsible for protection against both endogenous and exogenous oxidant toxicity (11). Interestingly Omega class and Beta class GSTs (such as Escherichia coli GST (EGST)) possess typical GRX activity toward widely used substrates, such as 2-hydroxyethyl disulfide (HEDS) (12–16). These GSTs have an active site cysteine, which is indispensable for GRX activity but not GST activity.The yeast prion protein Ure2 is composed of a disordered protease-sensitive N-terminal prion domain and a compact globular C-terminal domain, which shows high structural similarity to EGST (17). The C-terminal domain of Ure2 can be further structurally divided into two subdomains, the all-α-helix subdomain and the thioredoxin fold subdomain, which shows high structural homology to GRX. Ure2 is involved in the regulation of nitrogen metabolism and resistance to heavy metal ion toxicity (especially cadmium) and oxidative stress in S. cerevisiae (18, 19). In addition, Ure2 shows GPx activity toward both hydrogen peroxide and organic hydroperoxides such as cumene hydroperoxide and tert-butyl hydroperoxide (20). The discovery of the GPx activity of Ure2 (20) provides an explanation for its ability to protect yeast cells from oxidant toxicity (18). However, the reason that ure2Δ yeast cells are hypersensitive to cadmium remains unclear. In general, cadmium ions have a drastic effect on yeast cell growth, and the reasons are complicated. One possible reason for cadmium ion toxicity is that thioltransferases or GRXs can be inhibited by direct binding of cadmium to the two essential cysteine residues present in the thioltransferase active site (21). The inhibition of GRXs leads to complex effects on cell growth. Therefore, we used an in vitro assay to provide a system that allows detailed analysis of the activity of Ure2 and its relationship to that of GRX enzymes. Characterization of the allosteric behavior of the GRX activity of Ure2 revealed that Ure2 forms an active dimer within fibrils. In addition to providing information about the molecular structure of Ure2 fibrils, this also has implications for the molecular mechanism of Ure2 prion formation.     

Neurotoxic Mutants of the Prion Protein Induce Spontaneous Ionic Currents in Cultured Cells

The mechanisms by which prions kill neurons and the role of the cellular prion protein in this process are enigmatic. Insight into these questions is provided by the neurodegenerative phenotypes of transgenic mice expressing prion protein (PrP) molecules with deletions of conserved amino acids in the central region. We report here that expression in transfected cells of the most toxic of these PrP deletion mutants (Δ105–125) induces large, spontaneous ionic currents that can be detected by patch-clamping techniques. These currents are produced by relatively non-selective, cation-permeable channels or pores in the cell membrane and can be silenced by overexpression of wild-type PrP, as well as by treatment with a sulfated glycosaminoglycan. Similar currents are induced by PrP molecules carrying several different point mutations in the central region that cause familial prion diseases in humans. The ionic currents described here are distinct from those produced in artificial lipid membranes by synthetic peptides derived from the PrP sequence because they are induced by membrane-anchored forms of PrP that are synthesized by cells and that are found in vivo. Our results indicate that the neurotoxicity of some mutant forms of PrP is attributable to enhanced ion channel activity and that wild-type PrP possesses a channel-silencing activity. Drugs that block PrP-associated channels or pores may therefore represent novel therapeutic agents for treatment of patients with prion diseases.     

Elongation of Mouse Prion Protein Amyloid-Like Fibrils: Effect of Temperature and Denaturant Concentration

Prion protein is known to have the ability to adopt a pathogenic conformation, which seems to be the basis for protein-only infectivity. The infectivity is based on self-replication of this pathogenic prion structure. One of possible mechanisms for such replication is the elongation of amyloid-like fibrils.We measured elongation kinetics and thermodynamics of mouse prion amyloid-like fibrils at different guanidine hydrochloride (GuHCl) concentrations. Our data show that both increases in temperature and GuHCl concentration help unfold monomeric protein and thus accelerate elongation. Once the monomers are unfolded, further increases in temperature raise the rate of elongation, whereas the addition of GuHCl decreases it.We demonstrated a possible way to determine different activation energies of amyloid-like fibril elongation by using folded and unfolded protein molecules. This approach separates thermodynamic data for fibril-assisted monomer unfolding and for refolding and formation of amyloid-like structure.     

Synthetic Lipid Vesicles Recruit Native-Like Aggregates and Affect the Aggregation Process of the Prion Ure2p: Insights on Vesicle Permeabilization and Charge Selectivity

The yeast prion Ure2p polymerizes into native-like fibrils, retaining the overall structure and binding properties of the soluble protein. Recently we have shown that, similar to amyloid oligomers, the native-like Ure2p fibrils and their precursor oligomers are highly toxic to cultured mammalian cells when added to the culture medium, whereas Ure2p amyloid fibrils generated by heating the native-like fibrils are substantially harmless. We show here that, contrary to the nontoxic amyloid fibrils, the toxic, native-like Ure2p assemblies induce a significant calcein release from negatively charged phosphatidylserine vesicles. A minor and less-specific effect was observed with zwitterionic phosphatidylcholine vesicles, suggesting that the toxic aggregates preferentially bind to negatively charged sites on lipid membranes. We also found that cholesterol-enriched phospholipid membranes are protected against permeabilization by native-like Ure2p assemblies. Moreover, vesicle permeabilization appears charge-selective, allowing calcium, but not chloride, influx to be monitored. Finally, we found that the interaction with phosphatidylserine membranes speeds up Ure2p polymerization into oligomers and fibrils structurally and morphologically similar to the native-like Ure2p assemblies arising in free solution, although less cytotoxic. These data suggest that soluble Ure2p oligomers and native-like fibrils, but not amyloid fibrils, interact intimately with negatively charged lipid membranes, where they allow selective cation influx.     

Prion and Nonprion Amyloids: A Comparison Inspired by the Yeast Sup35 Protein

Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers. The [PSI⁺] determinant reflects polymerization of the Sup35 protein. Fragmentation of prion polymers by the Hsp104 chaperone represents a key step of the prion replication cycle. The frequency of fragmentation varies depending on the structure of the prion polymers and defines variation in the prion phenotypes, e.g., the suppressor strength of [PSI⁺] and stability of its inheritance. Besides [PSI⁺], overproduction of Sup35 can produce nonheritable phenotypically silent Sup35 amyloid-like polymers. These polymers are fragmented poorly and are present due to efficient seeding with the Rnq1 prion polymers, which occurs by several orders of magnitude more frequently than seeding of [PSI⁺] appearance. Such Sup35 polymers resemble human nonprion amyloids by their nonheritability, mode of appearance and increased size. Thus, a single protein, Sup35, can model both prion and nonprion amyloids. In yeast, these phenomena are distinguished by the frequency of polymer fragmentation. We argue that in mammals the fragmentation frequency also represents a key factor defining differing properties of prion and nonprion amyloids, including infectivity. By analogy with the Rnq1 seeding of nonheritable Sup35 polymers, the “species barrier” in prion transmission may be due to seeding by heterologous prion of nontransmissible type of amyloid, rather than due to the lack of seeding.Key Words: amyloid, prion, Rnq1, Sup35, Ure2, translation termination, yeast     

The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro

Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI ⁺] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI ⁺] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems.     

The Protein Phosphatase Inhibitors Okadaic Acid and Calyculin A Induce Apoptosis in Human Osteoblastic Cells

To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in osteoblastic cells, we examined the effects of okadaic acid (OA) and calyculin A (CA) on cultured human osteoblastic cells Saos-2 and MG63, and mouse osteoblastic MC3T3-E1 cells. After reaching confluence, these cells were exposed to varying concentrations of OA or CA. OA and CA induced cell death in all three cell lines in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were also observed in these cells by using the Hoechst 33342 stain. DNA ladder formation, a hallmark of apoptosis, was detected in Saos-2 and MG63 cells, but not in MC3T3-E1 cells by treatment of OA or CA. In the Saos-2 cells, OA- and CA-induced DNA ladder formation was dose-dependent with maximal effect at concentrations of 10 and 2 nM,respectively, and was time-dependent from 14 to 48 h. DNA ladder formation in response to OA and CA was revealed by using conventional ethidium bromide staining of electrophoresed DNA without using autoradiography. Beyond the maximal effects at the respective concentrations, however, cell death did not indicate DNA laddering, suggesting that phosphatase activity may be required for ladder formation. Our results indicate that apoptosis in the cultured osteoblastic cells is induced by moderate inhibition of PP-1 or PP-2A based on the known selectivity of okadaic acid and of calyculin A.     

ROS Production and Apoptosis Induction by Formation of Gts1p-Mediated Protein Aggregates

GTS1 of Saccharomyces cerevisiae is a pleiotropic gene. Its induction leads to a variety of biological phenomena represented by cell aggregation. The C-terminal polyglutamine sequence in Gts1p is indispensable for its pleiotropy and nuclear localization. This sequence is often observed in polyglutamine diseases, such as Huntington disease, and is believed to induce protein aggregation, leading to cell death. In this study, protein aggregates were formed in a polyglutamine-dependent manner in cells inducing GTS1, and heat-shock protein family, translation elongation factor, and mitochondrial proteins were trapped in Gts1p-mediated protein aggregates. Moreover, the polyglutamine sequence of Gts1p was indispensable to the induction of reactive oxygen species (ROS) production and apoptosis. Deletion of the genes encoding Por1p and Yhb1p altered the profiles of ROS production and apoptosis caused by GTS1 induction, suggesting that the trapping of these proteins in Gts1p-mediated protein aggregates inhibits the intrinsic functions of these proteins.     

The Saffold Virus-Penang 2B and 3C Proteins,but not the L Protein,Induce Apoptosis in HEp-2 and Vero Cells

Our previous work has shown that Saffold virus(SAFV) induced several rodent and primate cell lines to undergo apoptosis(Xu et al. in Emerg Microb Infect 3:1–8, 2014), but the essential viral proteins of SAFV involved in apoptotic activity lack study. In this study, we individually transfected the viral proteins of SAFV into HEp-2 and Vero cells to assess their ability to induce apoptosis, and found that the 2 B and 3 C proteins are proapoptotic. Further investigation indicated the transmembrane domain of the 2 B protein is essential for the apoptotic activity and tetramer formation of the 2 B protein. Our research provides clues for the possible mechanisms of apoptosis induced by SAFV in different cell lines. It also opens up new directions to study viral proteins(the 2 B, 3 C protein), and sets the stage for future exploration of any possible link between SAFV, inclusive of its related uncultivable genotypes, and multiple sclerosis.     

The N-Terminal Sequence of Prion Protein Consists an Epitope Specific to the Abnormal Isoform of Prion Protein (PrPSc)

The conformation of abnormal prion protein (PrP^Sc) differs from that of cellular prion protein (PrP^C), but the precise characteristics of PrP^Sc remain to be elucidated. To clarify the properties of native PrP^Sc, we attempted to generate novel PrP^Sc-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP^Sc purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP^Sc from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP^C from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31–39 and 41–47, respectively. This indicates that a PrP^Sc-specific epitope exists in the N-terminal region of PrP^Sc, and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP^Sc. We found that the ratio of proteinase K (PK)-sensitive PrP^Sc to PK-resistant PrP^Sc was constant throughout the disease time course.     

Concomitant Inhibition of MDM2 and Bcl-2 Protein Function Synergistically Induce Mitochondrial Apoptosis in AML

Disruption of Mdm2-p53 interaction activates p53 signaling, disrupts the balance ofantiapoptotic and proapoptotic Bcl-2 family proteins and induces apoptosis in acutemyeloid leukemia (AML). Overexpression of Bcl-2 may inhibit this effect. Thus,functional inactivation of antiapoptotic Bcl-2 proteins may enhance apoptogenic effects ofMdm2 inhibition. We here investigate the potential therapeutic utility of combinedtargeting of Mdm2 by Nutlin-3a and Bcl-2 by ABT-737, recently developed inhibitors ofprotein-protein interactions. Nutlin-3a and ABT-737 induced Bax conformational changeand mitochondrial apoptosis in AML cells in a strikingly synergistic fashion. Nutlin-3ainduced p53-mediated apoptosis predominantly in S and G₂/M cells, while cells in G₁ were protected through induction of p21. In contrast, ABT-737 induced apoptosis predominantly in G₁ , the cell cycle phase with the lowest Bcl-2 protein levels and Bcl-2/Bax ratios. In addition, Bcl-2 phosphorylation on Ser⁷⁰ was absent in G₁ but detectable in G₂/M, thus lower Bcl-2 levels and absence of Bcl-2 phosphorylation appeared to facilitate ABT-737-induced apoptosis of G₁ cells. The complementary effects of Nutlin-3a and ABT-737 in different cell cycle phases could, in part, account for their synergistic activity. Our data suggest that combined targeting of Mdm2 and Bcl-2 proteins could offer considerable therapeutic promise in AML.     

Prion Aggregates Are Recruited to the Insoluble Protein Deposit (IPOD) via Myosin 2-Based Vesicular Transport

Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed “Insoluble PrOtein Deposit (IPOD)”, but nothing is known about the mechanism of substrate recruitment to this site. The IPOD is located directly adjacent to the Phagophore Assembly Site (PAS) where the cell initiates autophagy and the Cytoplasm-to-Vacuole Targeting (CVT) pathway destined for delivery of precursor peptidases to the vacuole. Recruitment of CVT substrates to the PAS was proposed to occur via vesicular transport on Atg9 vesicles and requires an intact actin cytoskeleton and “SNAP (Soluble NSF Attachment Protein) Receptor Proteins (SNARE)” protein function. It is, however, unknown how this vesicular transport machinery is linked to the actin cytoskeleton. We demonstrate that recruitment of model amyloid PrD-GFP and the CVT substrate precursor-aminopeptidase 1 (preApe1) to the IPOD or PAS, respectively, is disturbed after genetic impairment of Myo2-based actin cable transport and SNARE protein function. Rather than accumulating at the respective deposition sites, both substrates reversibly accumulated often together in the same punctate structures. Components of the CVT vesicular transport machinery including Atg8 and Atg9 as well as Myo2 partially co-localized with the joint accumulations. Thus we propose a model where vesicles, loaded with preApe1 or PrD-GFP, are recruited to tropomyosin coated actin cables via the Myo2 motor protein for delivery to the PAS and IPOD, respectively. We discuss that deposition at the IPOD is not an integrated mandatory part of the degradation pathway for amyloid aggregates, but more likely stores excess aggregates until downstream degradation pathways have the capacity to turn them over after liberation by the Hsp104 disaggregation machinery.     

合成芪类化合物抑制肿瘤细胞增殖并诱导Bel-7402细胞凋亡

芪类化合物在癌的起始、促进和发展阶段均有化学抗癌活性,对其化学结构进行改造并进行作用机制研究,提高其抗肿瘤活性,有可能发现新的抗肿瘤药物.在合成23种芪类化合物的基础上,以MTT法测定其对9种肿瘤细胞系的抑制作用,发现多种化合物对SMMC-7721、BGC-823、HepG2、Bel-7402、MCF-7、SGC-7901细胞系具有较明显的抑制活性.以细胞周期分析、Hoechst 33258荧光染色及线粒体膜电位变化进行检测,发现经抑制作用较强的化合物HCQ-10处理后的 Bel-7402细胞发生G₂/M期阻滞,并继发细胞凋亡.实验结果说明,合成芪类化合物对SMMC-7721、BGC-823、HepG2、Bel-7402、MCF-7、SGC-7901细胞具有较为明显的增殖抑制作用,其抑制Bel 7402细胞增殖的作用机制为阻滞细胞于G₂/M期并诱导细胞继发凋亡.     

Plant Homologues of Components of MAPK (Mitogen-Activated Protein Kinase) Signal Pathways in Yeast and Animal Cells

As they respond to numerous extracellular and intracellularstimuli, plants develop various morphological features and thecapacity for a large variety of physiological processes duringtheir growth. If we are to understand the molecular basis ofsuch developments, we must elucidate the way in which signalsgenerated by such stimuli can be transduced into plant cellsand transmitted by cellular components to induce the appropriateterminal events. In yeast and animal systems, signal pathwaysthat are known collectively as MAPK (mitogen-activated proteinkinase) cascades have been shown to play a central role in thetransmission of various signals. The components of these pathwaysinclude the MAPK family, the activator kinases of the MAPK family(the MAPKK family) and the activator kinases of the MAPKK family(the MAPKKK family). The members of each respective family arestructurally conserved and signals are transmitted by similarphosphotransfer reactions at corresponding steps that are mediatedby a specific member of each family in turn. Both cDNAs andgenes that encode putative homologues of these components haverecently been isolated from plant sources. Some of them havebeen shown to be related not only structurally but also functionallyto members of the MAPK cascades of other organisms. These findingssuggest that plants have signal pathways that are analogousto the MAPK cascades in yeast and animal cells but it remainsto be proven that plant homologues do in fact constitute kinasecascades. Given the presence of so many homologues of MAPKsand MAPKKKs in a single plant species, namely, Arabidopsis thaliana,we can be fairly confident that the putative MAPK cascades areinvolved in various physiological processes in plants. (Received March 28, 1995; )     

Regulation of Lipid Signaling Pathways for Cell Survival and Apoptosis by bcl-2 in Prostate Carcinoma Cells

Compelling evidence indicates that activation of the JNK/SAPK signaling pathway is obligatory for apoptosis induction by multiple cell stresses that activate the sphingomyelin cycle. Moreover, ectopic expression of bcl-2 can impair apoptosis signaling by most of the cell stresses that activate the ceramide/JNK pathway. Here we show that enforced expression of bcl-2 protects prostate carcinoma cells against the induction of apoptosis by exogenous C₂-ceramide. Moreover, enforced bcl-2 expression blocked the capacity of C₂-ceramide to activate JNK1, indicating bcl-2 functions at the level of JNK1 or upstream of JNK1 in the ceramide/JNK pathway. The contribution of bcl-2 to the regulation of the arachidonate pathway for prostate carcinoma cell survival was also investigated using highly selective inhibitors of arachidonate metabolism. Our results indicate bcl-2 can protect cells against diminished availability of arachidonic acid, 12-HETE, and 15-HETE. Finally, arachidonic acid substantially suppresses the induction of apoptosis by C₂-ceramide, providing evidence for the opposing influences of these lipid signaling pathways in the mediation of prostate carcinoma cell survival. These results provide evidence for opposing influences of the ceramide and arachidonate signaling pathways in the mediation of cell death and cell survival, respectively, in prostate carcinoma cells and suggest a dual role for bcl-2 in this context.