Proteome Analysis of Peroxisomes from Etiolated Arabidopsis Seedlings Identifies a Peroxisomal Protease Involved in β-Oxidation and Development

Plant peroxisomes are highly dynamic organelles that mediate a suite of metabolic processes crucial to development. Peroxisomes in seeds/dark-grown seedlings and in photosynthetic tissues constitute two major subtypes of plant peroxisomes, which had been postulated to contain distinct primary biochemical properties. Multiple in-depth proteomic analyses had been performed on leaf peroxisomes, yet the major makeup of peroxisomes in seeds or dark-grown seedlings remained unclear. To compare the metabolic pathways of the two dominant plant peroxisomal subtypes and discover new peroxisomal proteins that function specifically during seed germination, we performed proteomic analysis of peroxisomes from etiolated Arabidopsis (Arabidopsis thaliana) seedlings. The detection of 77 peroxisomal proteins allowed us to perform comparative analysis with the peroxisomal proteome of green leaves, which revealed a large overlap between these two primary peroxisomal variants. Subcellular targeting analysis by fluorescence microscopy validated around 10 new peroxisomal proteins in Arabidopsis. Mutant analysis suggested the role of the cysteine protease RESPONSE TO DROUGHT21A-LIKE1 in β-oxidation, seed germination, and growth. This work provides a much-needed road map of a major type of plant peroxisome and has established a basis for future investigations of peroxisomal proteolytic processes to understand their roles in development and in plant interaction with the environment.Peroxisomes, originally known as microbodies, are small and single-membrane eukaryotic organelles that compartmentalize various oxidative metabolic functions. Most peroxisomal matrix proteins carry a C-terminal tripeptide named PEROXISOME TARGETING SIGNAL TYPE1 (PTS1), and fewer contain an N-terminal nonapeptide, PTS2 (Lanyon-Hogg et al., 2010). PTS1 is further divided into major and minor PTS1s. Major PTS1 tripeptides, such as SKL> and SRL> (> represents the stop codon), are by themselves sufficient to direct a protein to the peroxisome (Reumann, 2004), whereas minor PTS1s are usually found in low-abundance proteins and require additional upstream elements for peroxisomal targeting (Kaur et al., 2009). Peroxisomes are highly variable morphologically and metabolically, as their size, shape, abundance, and enzymatic content can differ depending on the species, tissue and cell type, and prevailing environmental conditions (Beevers, 1979; van den Bosch et al., 1992; Kaur et al., 2009; Hu et al., 2012; Schrader et al., 2012).Plant peroxisomes participate in a wide range of metabolic processes, such as lipid metabolism, photorespiration, detoxification, biosynthesis of jasmonic acid, and metabolism of indole-3-butyric acid (IBA), nitrogen, sulfite, and polyamine (Kaur et al., 2009; Hu et al., 2012). Specific names had been given to certain types of peroxisomes due to their unique metabolic properties. For example, the term glyoxysome was coined when a new type of organelle that contained enzymes of the glyoxylate cycle was identified from the endosperm of castor bean (Ricinus communis; Breidenbach et al., 1968). It was later realized that glyoxysomes are in fact a type of peroxisome, and Beevers (1979) subsequently classified plant peroxisomes into three subtypes based on their primary biochemical functions. Glyoxysomes are located in storage organs such as fatty seedling tissues and play a major role in converting fatty acids to sugar; leaf peroxisomes are involved in photorespiration; and nonspecialized peroxisomes exist in other plant tissues and perform unknown functions.The primary function of leaf peroxisomes is the recycling of phosphoglycolate during photorespiration, a process coordinated by chloroplasts, peroxisomes, mitochondria, and the cytosol. In this pathway, phosphoglycolate produced by the oxygenase activity of Rubisco is ultimately converted to glycerate, which reenters the chloroplastic Calvin-Benson cycle (Foyer et al., 2009; Peterhansel et al., 2010). The peroxisome-localized enzymes glycolate oxidase (GOX), catalase, aminotransferase (serine:glyoxylate aminotransferase [SGT] and glutamate-glyoxylate aminotransferase [GGT]), HYDROXYPYRUVATE REDUCTASE1 (HPR1), and peroxisomal malate dehydrogenase (PMDH) are involved in the process (Reumann and Weber, 2006). On the other hand, lipid mobilization through fatty acid β-oxidation and the glyoxylate cycle is the main function for peroxisomes in seeds and germinating seedlings. In this process, fatty acids are first activated into fatty acyl-CoA esters by the acyl-activating enzyme (AAE)/acyl-CoA synthetase before entering the β-oxidation cycle, during which an acetyl-CoA is cleaved in each cycle by the successive action of acyl-CoA oxidase (ACX), multifunctional protein (MFP), and 3-keto-acyl-CoA thiolase (KAT). Acetyl-CoA, an end product of β-oxidation, is further converted to four-carbon carbohydrates by the glyoxylate cycle, in which isocitrate lyase (ICL) and malate synthase (MLS) are two key enzymes that function exclusively in this pathway. Products of the glyoxylate cycle exit the peroxisome, enter gluconeogenesis, and are further converted to hexose and Suc to fuel the postgerminative development of seedlings (Penfield et al., 2006).Immunocytochemical studies of germinating seeds from pumpkin (Cucurbita pepo), watermelon (Citrullis vulgaris), and cucumber (Cucumis sativus) demonstrated that seed peroxisomes (glyoxysomes) are directly transformed into leaf peroxisomes during greening of the cotyledons without de novo biogenesis of leaf peroxisomes (Titus and Becker, 1985; Nishimura et al., 1986; Sautter, 1986). This conversion was illustrated by the import of photorespiratory enzymes and their concomitant presence with glyoxylate cycle enzymes within the same organelle. Furthermore, the increase in abundance of photorespiratory enzymes coincided with the marked decrease, and subsequently the absence, of glyoxylate cycle enzymes (ICL and/or MLS) at the culmination of this process (Titus and Becker, 1985; Nishimura et al., 1986; Sautter, 1986). It was suggested that the specific names for plant peroxisomal variants should be eliminated because protein composition between leaf peroxisomes and glyoxysomes may differ by only two proteins (i.e. ICL and MLS) out of the over 100 total proteins in the peroxisome (Pracharoenwattana and Smith, 2008). This prediction needed to be tested. In addition, mutants lacking core peroxisome biogenesis factors or major β-oxidation enzymes are nonviable, suggesting that peroxisomes are essential to embryogenesis and seed germination (Hu et al., 2012). However, how peroxisomes contribute to seed germination and seedling establishment is not completely understood. In the past, studies have been successfully undertaken to catalog the proteome of mitochondria and plastids isolated from different plant tissues, which uncovered unique facets of organelle metabolism in various tissues (van Wijk and Baginsky, 2011; Havelund et al., 2013; Lee et al., 2013). As such, it was necessary to establish a protein atlas for peroxisomes in dark-grown seedlings.Proteomic analyses of leaf peroxisomes and peroxisomes from suspension-cultured, leaf-derived cells followed by protein subcellular localization studies confirmed a total of over 30 new peroxisomal proteins, uncovering additional metabolic functions for leaf peroxisomes (Fukao et al., 2002; Reumann et al., 2007, 2009; Eubel et al., 2008; Babujee et al., 2010; Kataya and Reumann, 2010; Quan et al., 2010). For Arabidopsis (Arabidopsis thaliana), around 100 peroxisomal proteins were shown to be present in leaves or leaf-derived cells. Compared with the over 80 bona fide peroxisomal proteins detected by leaf peroxisomal proteomics (Reumann et al., 2007, 2009), the number of proteins identified from peroxisomal proteomic studies on etiolated seedlings was significantly smaller, with less than 10 known peroxisomal proteins from Arabidopsis (Fukao et al., 2003) and approximately 31 from soybean (Glycine max; Arai et al., 2008a, 2008b). Thus, a more in-depth analysis of the proteome of peroxisomes from these tissues was highly needed.Here, we performed proteomic analysis of peroxisomes isolated from etiolated Arabidopsis seedlings and detected peroxisomal proteins that encompass most of the known plant peroxisomal metabolic pathways. Fluorescence microscopy verified the peroxisomal localization of a number of proteins newly identified in this study or detected from previous proteomics that had not been verified by independent means. Reverse genetic analysis demonstrated the role for a Cys protease in germination, β-oxidation, and growth.     

The mRNA-stabilizing Factor HuR Protein Is Targeted by β-TrCP Protein for Degradation in Response to Glycolysis Inhibition

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase β-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, ²⁹⁶EEAMAIAS³⁰⁴, in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to β-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy.     

Activation of p107 by Fibroblast Growth Factor,Which Is Essential for Chondrocyte Cell Cycle Exit,Is Mediated by the Protein Phosphatase 2A/B55α Holoenzyme

The phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible cyclin-dependent kinases (CDKs) and serine/threonine protein phosphatase 2A (PP2A). Two trimeric holoenzymes consisting of the core PP2A catalytic/scaffold dimer and either the B55α or PR70 regulatory subunit have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55α levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55α has not been elucidated. Here we show that fibroblast growth factor 1 (FGF1), which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107-PP2A/B55α complexes coinciding with p107 dephosphorylation. Reciprocal solution-based mass spectrometric analysis identified the PP2A/B55α complex as a major component in p107 complexes, which also contain E2F/DPs, DREAM subunits, and/or cyclin/CDK complexes. Of note, p107 is one of the preferred partners of B55α, which also associates with pRB in RCS cells. FGF1-induced dephosphorylation of p107 results in its rapid accumulation in the nucleus and formation of larger complexes containing p107 and enhances its interaction with E2F4 and other p107 partners. Consistent with a key role of B55α in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55α results in marked dephosphorylation of p107 while B55α knockdown results in hyperphosphorylation. More importantly, knockdown of B55α dramatically delays FGF1-induced dephosphorylation of p107 and slows down cell cycle exit. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in early recruitment of p107 to the MYC promoter, an FGF1/E2F-regulated gene. Our results suggest a model in which FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylation for several hours after p107 dephosphorylation in maturing chondrocytes.     

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]_i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of −5 mV, the I_Ca,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser¹⁶ was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca²⁺ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.     

The Multifunctional Protein in Peroxisomal β-Oxidation: STRUCTURE AND SUBSTRATE SPECIFICITY OF THE ARABIDOPSIS THALIANA PROTEIN MFP2*

Plant fatty acids can be completely degraded within the peroxisomes. Fatty acid degradation plays a role in several plant processes including plant hormone synthesis and seed germination. Two multifunctional peroxisomal isozymes, MFP2 and AIM1, both with 2-trans-enoyl-CoA hydratase and l-3-hydroxyacyl-CoA dehydrogenase activities, function in mouse ear cress (Arabidopsis thaliana) peroxisomal β-oxidation, where fatty acids are degraded by the sequential removal of two carbon units. A deficiency in either of the two isozymes gives rise to a different phenotype; the biochemical and molecular background for these differences is not known. Structure determination of Arabidopsis MFP2 revealed that plant peroxisomal MFPs can be grouped into two families, as defined by a specific pattern of amino acid residues in the flexible loop of the acyl-binding pocket of the 2-trans-enoyl-CoA hydratase domain. This could explain the differences in substrate preferences and specific biological functions of the two isozymes. The in vitro substrate preference profiles illustrate that the Arabidopsis AIM1 hydratase has a preference for short chain acyl-CoAs compared with the Arabidopsis MFP2 hydratase. Remarkably, neither of the two was able to catabolize enoyl-CoA substrates longer than 14 carbon atoms efficiently, suggesting the existence of an uncharacterized long chain enoyl-CoA hydratase in Arabidopsis peroxisomes.     

Cross-talk between Serine/Threonine Protein Phosphatase 2A and Protein Tyrosine Phosphatase 1B Regulates Src Activation and Adhesion of Integrin αIIbβ3 to Fibrinogen

Integrin α_IIbβ₃ signaling mediated by kinases and phosphatases participate in hemostasis and thrombosis, in part, by supporting stable platelet adhesion. Our previous studies indicate that the genetic manipulation of PP2Acα (α isoform of the catalytic subunit of protein phosphatase 2A) negatively regulate the adhesion of human embryonal kidney 293 cells expressing α_IIbβ₃ to fibrinogen. Here, we demonstrated that small interference RNA (siRNA) mediated knockdown of PP2Acα in 293 α_IIbβ₃ cells led to the dephosphorylation of Src Tyr-529, phosphorylation of Src Tyr-418 and an increased Src kinase activity. Conversely, overexpression of PP2Acα decreased the basal Src activity. Pharmacological inhibition of PP2Ac in human platelets or PP2Acα knockdown in primary murine megakaryocytes resulted in Src activation. PP2Acα-depleted 293 α_IIbβ₃ cells did not alter the serine (Ser) phosphorylation of Src but enhanced the Ser-50 phosphorylation of protein tyrosine phosphatase 1B (PTP-1B) with a concomitant increase in the PTP-1B activity. Src activation in the PP2Acα-depleted 293 α_IIbβ₃ cells was abolished by siRNA mediated knockdown of PTP-1B. Pharmacological inhibition of Src or knockdown of Src, PTP-1B blocked the enhanced activation of extracellular signal-regulated kinase (ERK1/2) and the increased adhesiveness of PP2Acα-depleted 293 α_IIbβ₃ cells to fibrinogen, respectively. Thus, inactivation of PP2Acα promotes hyperphosphorylation of PTP-1B Ser-50, elevates PTP-1B activity, which dephosphorylates Src Tyr-529 to activate Src and its downstream ERK1/2 signaling pathways that regulate α_IIbβ₃ adhesion. Moreover, these studies extend the notion that a cross-talk between Ser/Thr and Tyr phosphatases can fine-tune α_IIbβ₃ outside-in signaling.     

Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

An understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungus Cryptococcus neoformans is important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth of C. neoformans on fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor for C. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity for C. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence in C. neoformans by multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.     

2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.     

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Peroxisomes play an essential role in a number of important metabolic pathways including β-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various β-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal β-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp²⁴⁹ to Arg²⁵¹) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp²⁴⁹ to Arg²⁵¹ and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.     

Protein Phosphatase 2A Isoforms Utilizing Aβ Scaffolds Regulate Differentiation through Control of Akt Protein

Protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. It consists of a scaffolding A subunit to which a catalytic C subunit and one of many regulatory B subunits bind. Of the more than 80 PP2A isoforms, 10% use Aβ as a scaffold. This study demonstrates the isoform-specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K, and p53 in transformation. Comparisons of polyoma and SV40 small T antigens implicate Aβ in the control of differentiation. Knockdown of Aβ enhanced differentiation. Akt signaling regulated differentiation; its activation or inhibition promoted or blocked it, respectively. Aβ bound Akt. Enhancement of PP2A Aβ/Akt interaction by polyoma small T antigen increased turnover of Akt Ser-473 phosphorylation. Conversely, knockdown of Aβ promoted Akt activity and reduced turnover of phosphate at Ser-473 of Akt. These data provide new insight into the regulation of Akt, a protein of extreme importance in cancer. Furthermore, our results suggest that the role for Aβ in differentiation and perhaps tumor suppression may lie partly in its ability to negatively regulate Akt.     

Protein β-Sheet Nucleation Is Driven by Local Modular Formation

Despite its central role in the protein folding process, the specific mechanism(s) behind β-sheet formation has yet to be determined. For example, whether the nucleation of β-sheets, often containing strands separated in sequence by many residues, is local or not remains hotly debated. Here, we investigate the initial nucleation step of β-sheet formation by performing an analysis of the smallest β-sheets in a non-redundant dataset on the grounds that the smallest sheets, having undergone little growth after nucleation, will be enriched for nucleating characteristics. We find that the residue propensities are similar for small and large β-sheets as are their interstrand pairing preferences, suggesting that nucleation is not primarily driven by specific residues or interacting pairs. Instead, an examination of the structural environments of the two-stranded sheets shows that virtually all of them are contained in single, compact structural modules, or when multiple modules are present, one or both of the chain termini are involved. We, therefore, find that β-nucleation is a local phenomenon resulting either from sequential or topological proximity. We propose that β-nucleation is a result of two opposite factors; that is, the relative rigidity of an associated folding module that holds two stretches of coil close together in topology coupled with sufficient chain flexibility that enables the stretches of coil to bring their backbones in close proximity. Our findings lend support to the hydrophobic zipper model of protein folding (Dill, K. A., Fiebig, K. M., and Chan, H. S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1942–1946). Implications for protein folding are discussed.     

Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. β-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired β-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid β-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA β-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and β subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA β-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA β-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA β-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA β-oxidation and inducing cytokine expression by association with MTP.     

Peroxisomal β-oxidation activities and γ-decalactone production by the yeast Yarrowia lipolytica

γ-Decalactone is a peachy aroma compound resulting from the peroxisomal β-oxidation of ricinoleic acid by yeasts. The expression levels of acyl-CoA oxidase (gene deletion) and 3-ketoacyl-CoA thiolase activities (gene amplification on replicative plasmids) were modified in the yeast Yarrowia lipolytica. The effects of these modifications on β-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall β-oxidation activity. The disruption of one of the acyl-CoA oxidase genes resulted in an enhanced activity. The enhancement led to an increase of overall β-oxidation activity but reduced the γ-decalactone production rates. This seemed to indicate a non-rate-limiting role for β-oxidation in the biotransformation of ricinoleic acid to γ-decalactone by the yeast Yarrowia lipolytica. All strains produced and then consumed γ-decalactone. We checked the ability of the different strains to consume γ-decalactone in a medium containing the lactone as sole carbon source. The consumption of the strain overexpressing acyl-CoA oxidase activity was higher than that of the wild-type strain. We␣concluded that peroxisomal β-oxidation is certainly involved in γ-decalactone catabolism by the yeast Y.␣lipolytica. The observed production rates probably depend on an equilibrium between production and consumption of the lactone. Received: 13 June 1997 / Received revision: 2 October 1997 / Accepted: 14 October 1997     

Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser⁴¹ of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca²⁺ release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser⁴¹. This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca²⁺ homeostasis.