A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote,Trichomonas vaginalis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localized in the cytosol of Trichomonas vaginalis, was partially purified. The enzyme is specific for NAD⁺ and is similar in most of its catalytic properties to glycolytic GAPDHs from other organisms. Its sensitivity to koningic acid is similar to levels observed in GAPDHs from eubacteria and two orders of magnitude lower than those observed for eukaryotic GAPDHs. The complete amino acid sequence of T. vaginalis GAPDH was derived from the N-terminal sequence of the purified protein and the deduced sequence of a cDNA clone. It showed great similarity to other eubacterial and eukaryotic GAPDH sequences. The sequence of the S-loop displayed a eubacterial signature. The overall sequence was more similar to eubacterial sequences than to cytosolic and glycosomal eukaryotic sequences. In phylogenetic trees obtained with distance matrix and parsimony methods T. vaginalis GAPDH clustered with its eubacterial homologs. GAPDHs of other amitochondriate protists, belonging to early branches of the eukaryotic lineage (Giardia lamblia and Entamoeba histolytica—Smith M.W. and Doolittle R.F., unpublished data in GenBank), showed typical eukaryotic signatures and clustered with other eukaryotic sequences, indicating that T. vaginalis GAPDH occupies an anomalous position, possibly due to horizontal gene transfer from a eubacterium. Correspondence to: M. Müller     

An Uncoupling of the Processes of Oxidation and Phosphorylation in Glycolysis

Data on alterations of the properties of glyceraldehyde-3-phosphate dehydrogenase upon oxidation of its functional groups are reviewed; a mechanism of uncoupling of oxidation and phosphorylation in glycolysis is considered. Possible ways of regulating uncoupling, and the physiological importance of this process, are discussed.     

Moonlighting glyceraldehyde-3-phosphate dehydrogenase: posttranslational modification,protein and nucleic acid interactions in normal cells and in human pathology

Abstract

Moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) exhibits multiple functions separate and distinct from its historic role in energy production. Further, it exhibits dynamic changes in its subcellular localization which is an a priori requirement for its multiple activities. Separately, moonlighting GAPDH may function in the pathology of human disease, involved in tumorigenesis, diabetes, and age-related neurodegenerative disorders. It is suggested that moonlighting GAPDH function may be related to specific modifications of its protein structure as well as the formation of GAPDH protein: protein or GAPDH protein: nucleic acid complexes.     

Inactivation of cellular enzymes by carbonyls and protein-bound glycation/glycoxidation products

Diabetic plasma contains elevated levels of glucose and various low-molecular-weight carbonyl compounds derived from the metabolism of glucose and related materials. These compounds react with protein side chains (Arg, Lys, Cys, and His) to give glycated materials and advanced glycation end products. In this study, we have examined the effect of glucose and carbonyl compounds (methylglyoxal, glyoxal, glycolaldehyde, and hydroxyacetone), and glycation products arising from reaction of these materials with model proteins, on the activity of three key cellular enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutathione reductase, and lactate dehydrogenase, both in isolation and in cell lysates. In contrast to glucose (1M, both fresh and aged for 8 weeks), which had no effect, marked inhibition of all three enzymes was observed with methylglyoxal and glyoxal. GAPDH was also inhibited by glycolaldehyde and hydroxyacetone. Incubation of these enzymes with proteins that had been preglycated with methylglyoxal, but not glucose, also resulted in significant time- and concentration-dependent inhibition with both isolated enzymes and cell lysates. This inhibition was not metal ion, oxygen, superoxide dismutase, or catalase dependent, suggesting that inhibition is not radical mediated. These effects are suggested to be due to direct adduction of the free- or protein-bound carbonyls with the target enzyme. Such an interpretation is supported by the detection of the loss of thiol groups on GAPDH and the detection of cross-linked materials on protein gels. Though direct comparison of the extent of inhibition induced by free versus protein-bound carbonyls was not possible, the significantly higher concentrations of the latter materials over the former in diabetic plasma and cells lead us to suggest that alterations in the activity of key cellular enzymes induced by glycated proteins may play a significant role in the development of diabetic complications.     

Starvation and temperature upshift cause an increase in the enzymatically active cell wall-associated glyceraldehyde-3-phosphate dehydrogenase protein in yeast

The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase (cwGAPDH) activity in Saccharomyces cerevisiae increases (two- to 10-fold, depending on the strain) in response to starvation and temperature upshift. Assays using transformants carrying pTDH, a yeast centromer derivative plasmid containing the Candida albicans TDH3 gene (encoding GAPDH) fused in frame with the yeast SUC2-coding region for internal invertase, showed that starvation and/or temperature upshift result in a similar increase in both cwGAPDH and cell wall-associated invertase activities. In addition, this incorporation of GAPDH protein into the cell wall in response to stress does not require (i) de novo protein synthesis, indicating that preexisting cytosolic enzyme is incorporated into the cell wall, (ii) nor the participation of the ubiquitin yeast stress response system, as no differences were observed between wild-type and polyubiquitin-depleted (Deltaubi4) strains.     

Mapping quantitative trait loci for milling quality, protein content and color characteristics of rice using a recombinant inbred line population derived from an elite rice hybrid

Milling properties, protein content, and flour color are important factors in rice. A marker-based genetic analysis of these traits was carried out in this study using recombinant inbred lines (RILs) derived from an elite hybrid cross ’Shanyou 63’, the most-widely grown rice hybrid in production in China. Correlation analysis shows that the traits were inter-correlated, though the coefficients were generally small. Quantitative trait locus (QTL) analysis with both interval mapping (IM) and composite interval mapping (CIM) revealed that the milling properties were controlled by the same few loci that are responsible for grain shape. The QTL located in the interval of RM42-C734b was the major locus for brown rice yield, and the QTL located in the interval of C1087-RZ403 was the major locus for head rice yield. These two QTLs are the loci for grain width and length, respectively. The Wx gene plays a major role in determining protein content and flour color, and is modified by several QTLs with minor effect. The implications of the results in rice breeding were discussed. Received: 15 September 2000 / Accepted: 31 March 2001     

Molecular cloning,sequence analysis and tissue-specific expression of Akirin2 gene in Tianfu goat

The Akirin2 gene is a nuclear factor and is considered as a potential functional candidate gene for meat quality. To better understand the structures and functions of Akirin2 gene, the cDNA of the Tianfu goat Akirin2 gene was cloned. Sequence analysis showed that the Tianfu goat Akirin2 cDNA full coding sequence (CDS) contains 579 bp nucleotides that encode 192 amino acids. A phylogenic tree of the Akirin2 protein sequence from the Tianfu goat and other species revealed that the Tianfu goat Akirin2 was closely related with cattle and sheep Akirin2. RT-qPCR analysis showed that Akirin2 was expressed in the myocardium, liver, spleen, lung, kidney, leg muscle, abdominal muscle and the longissimus dorsi muscle. Especially, high expression levels of Akirin2 were detected in the spleen, lung, and kidney whereas lower expression levels were seen in the liver, myocardium, leg muscle, abdominal muscle and longissimus dorsi muscle. Temporal mRNA expression showed that Akirin2 expression levels in the longissimus dorsi muscle, first increased then decreased from day 1 to month 12. Western blotting results showed that the Akirin2 protein was only detected in the lung and three skeletal muscle tissues.     

A novel role for fatty acid transport protein 1 in the regulation of tricarboxylic acid cycle and mitochondrial function in 3T3-L1 adipocytes

Fatty acid transport proteins (FATPs) are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. Whereas some FATP1 translocates to the plasma membrane in response to insulin, the majority of FATP1 remains within intracellular structures and bioinformatic and immunofluorescence analysis of FATP1 suggests the protein primarily resides in the mitochondrion. To evaluate potential roles for FATP1 in mitochondrial metabolism, we used a proteomic approach following immunoprecipitation of endogenous FATP1 from 3T3-L1 adipocytes and identified mitochondrial 2-oxoglutarate dehydrogenase. To assess the functional consequence of the interaction, purified FATP1 was reconstituted into phospholipid-containing vesicles and its effect on 2-oxoglutarate dehydrogenase activity evaluated. FATP1 enhanced the activity of 2-oxoglutarate dehydrogenase independently of its acyl-CoA synthetase activity whereas silencing of FATP1 in 3T3-L1 adipocytes resulted in decreased activity of 2-oxoglutarate dehydrogenase. FATP1 silenced 3T3-L1 adipocytes exhibited decreased tricarboxylic acid cycle activity, increased cellular NAD⁺/NADH, increased fatty acid oxidation, and increased lactate production indicative of altered mitochondrial energy metabolism. These results reveal a novel role for FATP1 as a regulator of tricarboxylic acid cycle activity and mitochondrial function.     

Mapping of two genes for resistance to clubroot (Plasmodiophora brassicae) in a population of doubled haploid lines of Brassica oleracea by means of RFLP and AFLP markers

A genetic map covering 615 cM in 12 linkage groups was assembled based on 92 RFLP and AFLP markers segregating in a population of 107 doubled haploid lines (DH lines) of Brassica oleracea. The DH-line population was obtained through microspore culture from the of two homozygous parents: DH-line Bi derived from the cabbage landrace Bindsachsener, and DH-line Gr from broccoli cv ‘Greenia’. Sixty-five percent of the loci, and in some cases complete linkage groups, displayed distorted segregation ratios, a frequency much higher than that observed in populations of the same species. DH-line Bi was resistant to clubroot, which is caused by a Dutch field isolate of Plasmodiophora brassicae. Resistance in the DH-line population was determined in two ways: by assigning symptom grades to each plant, and by measuring the fresh weights of the healthy and affected parts of the root system of each plant. Using a multiple QTL mapping approach to analyze the fresh weight data, we found two loci for clubroot resistance; these were designated pb-3 and pb-4. The additive effects of these loci were responsible for 68% of the difference between the parents and for 60% of the genetic variance among DH-line means. Also, indications for the presence of two additional, minor QTLs were found. Analysis of symptom grades revealed the two QTLs pb-3 and pb-4, as well as one of the two minor QTLs indicated by analysis of the fresh weight data. Received: 29 April 1996 / Accepted: 10 May 1996     

Role of the glycolytic protein,glyceraldehyde-3-phosphate dehydrogenase,in normal cell function and in cell pathology

The glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be an archtypical protein of limited excitement. However, independent studies from a number of different laboratories reported a variety of diverse biological properties of the GAPDH protein. As a membrane protein, GAPDH functions in endocytosis; in the cytoplasm, it is involved in the translational control of gene expression; in the nucleus, it functions in nuclear tRNA export, in DNA replication, and in DNA repair. The intracellular localization of GAPDH may be dependent on the proliferative state of the cell. Recent studies identified a role for GAPDH in neuronal apoptosis. GAPDH gene expression was specifically increased during programmed neuronal cell death. Transfection of neuronal cells with antisense GAPDH sequences inhibited apoptosis. Lastly, GAPDH may be directly involved in the cellular phenotype of human neurodegenerative disorders, especially those characterized at the molecular level by the expansion of CAG repeats. In this review, the current status of ongoing GAPDH studies are described (with the exception of its unique oxidative modification by nitric oxide). Consideration of future directions are suggested. J. Cell. Biochem. 66:133-140, 1997. © 1997 Wiley-Liss, Inc.     

Expression analysis of ATAD3 isoforms in rodent and human cell lines and tissues

ATAD3 (ATPase family AAA-Domain containing protein 3) is a mitochondrial inner membrane ATPase with unknown but vital functions. Initial researches have focused essentially on the major p66-ATAD3 isoform, but other proteins and mRNAs are described in the data banks. Using a set of anti-peptide antibodies and by the use of rodent and human cell lines and organs, we tried to detail ATAD3 gene expression profiles and to verify the existence of the various ATAD3 isoforms. In rodent, the single ATAD3 gene is expressed as a major isoform of 67 kDa, (ATAD3l; long), in all cells and organs studied. A second isoform, p57-ATAD3s (small), is expressed specifically throughout brain development and in adult, and overexpressed around the peri-natal period. p57-ATAD3s is also expressed in neuronal and glial rodent cell lines, and during in vitro differentiation of primary cultured rat oligodendrocytes. Other smaller isoforms were also detected in a tissue-specific manner. In human and primates, ATAD3 paralogues are encoded by three genes (ATAD3A, 3B and 3C), each of them presenting several putative variants. Analyzing the expression of ATAD3A and ATAD3B with four specific anti-peptide antibodies, and comparing their expressions with in vitro expressed ATAD3 cDNAs, we were able to observe and define five isoforms. In particular, the previously described p72-ATAD3B is confirmed to be in certain cases a phosphorylated form of ATAD3As. Moreover, we observed that the ATAD3As phosphorylation level is regulated by insulin and serum. Finally, exploring ATAD3 mRNA expression, we confirmed the existence of an alternative splicing in rodent and of several mRNA isoforms in human.     

Bifunctional apoptosis regulator (BAR), an endoplasmic reticulum (ER)-associated E3 ubiquitin ligase, modulates BI-1 protein stability and function in ER Stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.     

Associations of growth hormone secretagogue receptor (GHSR) genes polymorphisms and protein structure changes with carcass traits in sheep

Growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor that binds ghrelin, plays an important role in the central regulation of pituitary growth hormone secretion, food intake, and energy homeostasis. Ghrelin receptor (GHSR) modulates many physiological effects and therefore is a candidate gene for sheep production performance. Polymorphism of the GHSR gene was detected by PCR-SSCP and DNA sequencing methods in 463 individuals. Two different structures in protein and nine single nucleotide polymorphisms (SNPs) were identified. The evaluation of the associations between these SSCP patterns with carcass traits suggests a positive effect of genotype TT and B structure on carcass weight, and body length (P<0.05). In addition, the animal with TC had greater abdominal fat than those with TT and CC (P<0.05) while CC genotype contributed to low blood cholesterol (P=0.04). The results confirm the hints suggesting that GHSR is a preferential target for further investigation on mutations that influence carcass trait variations.     

Differential expression of genes and changes in glucose metabolism in the liver of liver-specific glucokinase gene knockout mice

To investigate the role of liver-specific expression of glucokinase (GCK) in the pathogenesis of hyperglycemia and to identify candidate genes involved in mechanisms of the onset and progression of maturity onset diabetes of the young, type 2 (MODY-2), we examined changes in biochemical parameters and gene expression in GCK knockout (gck^w/–) and wild-type (gck^w/w) mice as they aged. Fasting blood glucose levels were found to be significantly higher in the gck^w/– mice, compared to age-matched gck^w/w mice, at all ages (P < 0.05), except at 2 weeks. GCK activity of gck^w/– mice was about 50% of that of wild type (gck^w/w) mice (P < 0.05). Glycogen content at 4 and 40 weeks of age was lower in gck^w/– mice compared to gck^w/w mice. Differentially expressed genes in the livers of 2 and 26 week-old liver-specific GCK knockout (gck^w/–) mice were identified by suppression subtractive hybridization (SSH), which resulted in the identification of phosphoenolpyruvatecarboxykinase (PEPCK, also called PCK1) and Sterol O-acyltransferase 2 (SOAT2) as candidate genes involved in pathogenesis. The expressions of PEPCK and SOAT2 along with glycogen phosphorylase (GP) and glycogen synthase (GS) were then examined in GCK knockout (gck^w/–) and wild-type (gck^w/w) mice at different ages. Changes in PEPCK mRNA levels were confirmed by real-time RT-PCR, while no differences in the levels of expression of SOAT2 or GS were observed in age-matched GCK knockout (gck^w/–) and wild-type (gck^w/w) mice. GP mRNA levels were decreased in 40-week old gck^w/– mice compared to age-matched gck^w/w mice. Changes in gluconeogenesis, delayed development of GCK and impaired hepatic glycogen synthesis in the liver potentially lead to the onset and progression of MODY2.     

Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.)

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.     

Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors

PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCF(βTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers.     

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q < 0.05; enrichment range 1.40–9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting β-cells and neurons and underline the existence of trans?nosology pathways in diabetes and its co-morbidities.     

Interaction of TPPP/p25 protein with glyceraldehyde-3-phosphate dehydrogenase and their co-localization in Lewy bodies

TPPP/p25, a flexible unstructured protein, binds to tubulin and induces aberrant microtubule assemblies. We identified hereby glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a new interacting partner of TPPP/p25. The immunoprecipitation and affinity chromatographic experiments with bovine brain cell-free extract revealed that the interaction was salt and NAD(+) sensitive while ELISA showed resistant and firm association of the two isolated proteins. In transfected HeLa cells at low expression level of EGFP-TPPP/p25, while the green fusion protein aligned at the microtubular network, GAPDH distributed uniformly in the cytosol. However, at high expression level, GAPDH co-localized with TPPP/p25 in the aggresome-like aggregate. Immunohistochemistry showed enrichment of TPPP/p25 and GAPDH within the alpha-synuclein positive Lewy body.     

Comparative analysis of caveolins in mouse and tammar wallaby: Role in regulating mammary gland function

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70–80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.     

Bio-informatics based analysis of genes implicated in alcohol mediated liver injury

Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.