Plants synthesize a sulfur-containing lipid, sulfoquinovosyldiacylglycerol, which is one of three nonphosphorus glycerolipids that provide the bulk of the structural lipids in photosynthetic membranes. Here, the identification of a novel gene, UDP-glucose pyrophosphorylase3 (UGP3), required for sulfolipid biosynthesis is described. Transcriptome coexpression analysis demonstrated highly correlated expression of UGP3 with known genes for sulfolipid biosynthesis in Arabidopsis thaliana. Liquid chromatography-mass spectrometry analysis of leaf lipids in two Arabidopsis ugp3 mutants revealed that no sulfolipid was accumulated in these mutants, indicating the participation of UGP3 in sulfolipid biosynthesis. From the deduced amino acid sequence, UGP3 was presumed to be a UDP-glucose pyrophosphorylase (UGPase) involved in the generation of UDP-glucose, serving as the precursor of the polar head of sulfolipid. Recombinant UGP3 was able to catalyze the formation of UDP-glucose from glucose-1-phosphate and UTP. A transient assay using fluorescence fusion proteins and UGPase activity in isolated chloroplasts indicated chloroplastic localization of UGP3. The transcription level of UGP3 was increased by phosphate starvation. A comparative genomics study on UGP3 homologs across different plant species suggested the structural and functional conservation of the proteins and, thus, a committing role for UGP3 in sulfolipid synthesis.

Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of ␣-synuclein. Although certain post-translational modifications of ␣-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of ␣-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50 -60% of the LBs that show an intense halo pattern resembling that of ␣-synuclein. By utilizing a cellular model of ␣-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of ␣-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of ␣-synuclein. Finally, we show that Pin1 binds synphilin-1, an ␣-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with ␣-synuclein, thus likely facilitating the formation of ␣-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for ␣-synuclein aggregation and in the resultant Lewy body formations in PD. Parkinson disease (PD)2 is one of the most common neurodegenerative disorders and is characterized by the loss of dopaminergic neurons in the substantia nigra and by the presence of cytoplasmic inclusions known as Lewy bodies (LBs) in surviving neurons (1, 2). LBs have classically been considered as a pathological hallmark of PD, consisting of many components, including ␣-synuclein, which is one of the major constituents (3, 4). The first indication of a pathogenic role for ␣-synuclein in PD came from the results of linkage analysis of mutations in its gene in autosomal dominant forms of the disease (5, 6). ␣-Synuclein is an unfolded protein in its native state, but in a pathological state it can be induced to form either ␣-helical or ␤-sheet structures that result in the formation of insoluble ␣-synuclein aggregates (7,8). The aggregation of ␣-synuclein can be modified by a range of factors, both in vitro and in vivo, including environmental regulators of pH, temperature, ionic strength, and oxidative stress and by intrinsic intracellular pathways (8). The latter of these regulatory networks includes several ␣-synuclein-binding proteins such as synphilin-1 (9) and posttranslational modifications of related molecules, such as phosphorylation and ubiquitination (10 -12). Synphilin-1 was identified as a protein that interacts with ␣-synuclein and has been shown t...

Arabidopsis transcription factors, MYB28, MYB29 and MYB76, positively regulate aliphatic glucosinolate (AGSL) biosynthesis. Mutual transcriptional regulation among these MYB genes makes it difficult to elucidate their individual function simply by analyzing knock-out mutants or ectopically overexpressing lines of these genes. In this study, we constructed transgenic lines expressing each MYB gene driven by its own promoter in the myb28myb29 background, where the expression of the endogenous MYB28, MYB29 and MYB76 was repressed with no AGSL accumulation. In leaves, transgenic MYB28 expression activated AGSL biosynthetic genes and restored accumulation of AGSLs with short side chains. Transgenic MYB29 expression activated the same biosynthetic pathway, but induction of the genes involved in side chain elongation was weaker than that by MYB28, resulting in a weaker recovery of AGSLs. Neither MYB28 nor MYB29 recovered long-chain AGSL accumulation. MYB76 was considered to require both MYB28 and MYB29 for its normal level of expression in leaves, and could not activate AGSL biosynthesis on its own. Interestingly, the accumulation in seeds of long- and short-chain AGSLs was restored by transgenic expression of MYB28 and MYB76, respectively. A sulfur stress experiment indicated that MYB28 expression was induced by sulfur deficiency, while the expression levels of MYB29 and MYB76 were positively correlated with sulfur concentration. This study illustrated how the individual MYBs work in regulating AGSL biosynthesis when expressed alone under normal transcriptional regulation.

The death-associated protein Daxx is a multifunctional factor that regulates a variety of cellular processes, including transcription and apoptosis. Several previous reports have indicated that Daxx is induced upon oxidative stress and is then subjected to phosphorylation-based functional modification. However, the precise molecular events underlying these phosphorylation events remain largely unknown. We report in our current study that the peptidyl-prolyl isomerase Pin1 is highly overexpressed in malignant human gliomas and inhibits Daxx-mediated cellular apoptosis. The targeted inhibition of Pin1 by small interfering RNA in A172 glioblastoma cells significantly enhances the apoptotic response induced by hydrogen peroxide or stimulatory Fas antibodies. This is in turn accompanied by the increased induction of Daxx and the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase pathway. Furthermore, Pin1 binds to the phosphorylated Ser 178 -Pro motif in the Daxx protein, and Pin1 overexpression results in the rapid degradation of Daxx via the ubiquitin-proteasome pathway. Moreover, a Daxx-S178A mutant, which cannot interact with Pin1, demonstrates higher proapoptotic activity and is refractory to Pin1-mediated antiapoptotic effects. We further found that the expression levels of Pin1 inversely correlate with the degree of Daxx nuclear accumulation in human glioblastoma tissues. These results together indicate that Pin1-mediated prolyl isomerization plays an important role in the negative regulation of Daxx and thereby inhibits the oxidative stress-induced cellular apoptotic response, particularly in malignant tumor cells where Pin1 is often overexpressed.

Amino acid and glucosinolate biosynthesis are two interdependent pathways; amino acid synthesis as a part of primary metabolism provides the precursors for glucosinolate biosynthesis in secondary metabolism. In our previous studies, the combination of coexpression analysis and metabolite profiling led to the identification of genes and key regulators involved in glucosinolate biosynthesis. Moreover, the integration of transcriptome and metabolome data of sulphur-deprived Arabidopsis plants revealed coordinate changes in the expression profiles of genes involved in glucosinolate and amino acid metabolism.This review provides an overview of our recent studies involving Arabidopsis mutant plants that exhibit impairment in the side-chain elongation process occurring during aliphatic glucosinolate biosynthesis by means of coexpression analysis and a novel metabolite profiling approach based on ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry (UPLC-TQMS) (Sawada et al. 2009a). Thus, this review highlights the advantages of the omics-based approach in identifying genes involved in glucosinolate biosynthesis.