Physiological roles of the beta-substituted alanine synthase gene family in Arabidopsis

The beta-substituted alanine (Ala) synthase (Bsas) family in the large superfamily of pyridoxal 5'-phosphate-dependent enzymes comprises cysteine (Cys) synthase (CSase) [O-acetyl-serine (thiol) lyase] and beta-cyano-Ala synthase (CASase) in plants. Nine genomic sequences encode putative Bsas proteins in Arabidopsis thaliana. The physiological roles of these Bsas isoforms in vivo were investigated by the characterization of T-DNA insertion mutants. Analyses of gene expression, activities of CSase and CASase, and levels of Cys and glutathione in the bsas mutants indicated that cytosolic Bsas1;1, plastidic Bsas2;1, and mitochondrial Bsas2;2 play major roles in Cys biosynthesis. Cytosolic Bsas1;1 has the most dominant contribution both in leaf and root, and mitochondrial Bsas2;2 plays a significant role in root. Mitochondrial Bsas3;1 is a genuine CASase. Nontargeted metabolome analyses of knockout mutants were carried out by a combination of gas chromatography time-of-flight mass spectrometry and capillary electrophoresis time-of-flight mass spectrometry. The level of gamma-glutamyl-beta-cyano-Ala decreased in the mutant bsas3;1, indicating the crucial role of Bsas3;1 in beta-cyano-Ala metabolism in vivo.     

Preferred substrate sequences for transglutaminase 2: screening using a phage-displayed peptide library

A large number of substrate proteins for tissue transglutaminase (TGase 2) have been identified in vivo and in vitro. Preference in primary sequence or secondary structure around the reactive glutamine residues in the substrate governs the reactivity for TGase 2. We established a screening system to identify preferable sequence as a glutamine-donor substrate using a phage-displayed peptide library. The results showed that several peptide sequences have higher reactivity and specificity to TGase 2 than those of preferable sequences previously reported. By analysis of the most reactive 12-amino acid sequence, T26 (HQSYVDPWMLDH), residues crucial to the enzymatic reaction were investigated. The following review summarizes the screening system and also the preference in substrate sequences that were obtained by this method and those previously reported.     

Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives

It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response in Arabidopsis

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.     

Auxin Response in Arabidopsis under Cold Stress: Underlying Molecular Mechanisms

To understand the mechanistic basis of cold temperature stress and the role of the auxin response, we characterized root growth and gravity response of Arabidopsis thaliana after cold stress, finding that 8 to 12 h at 4°C inhibited root growth and gravity response by ∼50%. The auxin-signaling mutants axr1 and tir1, which show a reduced gravity response, responded to cold treatment like the wild type, suggesting that cold stress affects auxin transport rather than auxin signaling. Consistently, expression analyses of an auxin-responsive marker, IAA2-GUS, and a direct transport assay confirmed that cold inhibits root basipetal (shootward) auxin transport. Microscopy of living cells revealed that trafficking of the auxin efflux carrier PIN2, which acts in basipetal auxin transport, was dramatically reduced by cold. The lateral relocalization of PIN3, which has been suggested to mediate the early phase of root gravity response, was also inhibited by cold stress. Additionally, cold differentially affected various protein trafficking pathways. Furthermore, the inhibition of protein trafficking by cold is independent of cellular actin organization and membrane fluidity. Taken together, these results suggest that the effect of cold stress on auxin is linked to the inhibition of intracellular trafficking of auxin efflux carriers.     

Alcadein Cleavages by Amyloid ��-Precursor Protein (APP) ��- and ��-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related ��-Secretase Dysfunction

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc_α, Alc_β, and Alc_γ. The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP α-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent γ-secretase complex, thereby generating “APP p3-like” and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc_α, p3-Alc_β, and p3-Alc_γ, whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor β-amyloid species Aβ42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc_α, p3-Alc_β, and p3-Alc_γ were not equivalent, suggesting that one type of γ-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect γ-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.     

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII−/− transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.