Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos

We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins.     

Identification of major Ca(2+)/calmodulin-dependent protein kinase phosphatase-binding proteins in brain: biochemical analysis of the interaction

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP-GAPDH complex by dephosphorylating and deactivating CaMKs that are responsible for the phosphorylation of GAPDH.     

Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells

In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples.     

Rat Brain Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with the Recombinant Cytoplasmic Domain of Alzheimer's β-Amyloid Precursor Protein

Abstract: Abundant senile plaques are a histological hallmark in the brain of Alzheimer's disease patients. Such plaques consist of, among many other constituents, aggregated βA4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimer's disease. To study possible interactions of brain proteins with 0A4 amyloid or other fragments of APP, βA4 amyloid and βA4 amyloid extended to the C-terminus of APP were recombinantly produced as fusion proteins termed "Amy" and "AmyC," respectively. Using Amy and AmyC affinity chromatography, a 35-kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C-terminus of APP. This 35-kDa protein was identified as the glycolytic enzyme gIyceraldehyde-3-phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the V_max of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.     

S-nitrosylation and S-glutathionylation of GAPDH: Similarities,differences, and relationships

The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation.Incubation of HEK 293 T cells in the presence of H₂O₂ or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H₂O₂ or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.     

基于核酸定量的核质分离质控方案

目的:拟建立一种方便快捷、经济有效的细胞核质分离鉴定方法。方法:本研究从DNA水平进行核质分离鉴定,选择GAPDH、ND1分别作为细胞核和细胞质标志基因,并根据GAPDH及ND1序列保守区设计引物,基于荧光定量PCR方法定性检测核质分离的效果。随后将本鉴定方法应用于其他种类细胞(BEAS-2B细胞、GT1-7细胞)及组织(小鼠心脏、肝脏、大脑)的核质分离鉴定。结果:在293T细胞应用该鉴定方法鉴定核质分离效果,结果显示:GAPDH、ND1在核组、质组中的含量存在明显差异,其中核标志基因GAPDH在细胞核中的比例达到了95%以上,质标志基因ND1在细胞质中的比例也达到了90%左右,这与从RNA水平及蛋白水平鉴定核质分离的结果一致。在其他种类的细胞(BEAS-2B细胞、GT1-7细胞)及组织(小鼠心脏、肝脏、大脑)应用该方法结果显示:细胞核组分中质标志基因ND1含量比293T细胞的有所增加,但仍可以实现核质分离鉴定。结论:本研究所建立的核质分离质控方法可以实现从DNA水平进行核质分离的鉴定,该方法更加经济、快捷。     

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke

In addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke.     

GAPDH is conformationally and functionally altered in association with oxidative stress in mouse models of amyotrophic lateral sclerosis

It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography-tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.     

Microtubule-associated protein 1B binds glyceraldehyde-3-phosphate dehydrogenase

Microtubule-associated protein 1B, MAP1B, is a major cytoskeletal protein during brain development and one of the largest brain MAPs associated with microtubules and microfilaments. Here, we identified several proteins that bind to MAP1B via immunoprecipitation with a MAP1B-specific antibody, by one and two-dimensional gel electrophoresis and subsequent mass spectrometry identification of precipitated proteins. In addition to tubulin and actin, a variety of proteins were identified. Among these proteins were glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 8, dihydropyrimidinase related proteins 2 and 3, protein-L-isoaspartate O-methyltransferase, beta-spectrin, and clathrin protein MKIAA0034, linking either directly or indirectly to MAP1B. In particular, GAPDH, a key glycolytic enzyme, was bound in large quantity to the heavy chain of MAP1B in adult brain tissue. In vitro binding studies confirmed a direct binding of GAPDH to MAP1B. In PC12 cells, GAPDH was found in cytoplasm and nuclei and partially co-localized with MAP1B. It disappeared from the cytoplasm under oxidative stress or after a disruption of cytoskeletal elements after colcemid or cytochalasin exposure. GAPDH may be essential in the local energy provision of cytoskeletal structures and MAP1B may help to keep this key enzyme close to the cytoskeleton.     

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis

Treatment with cytosine beta-D-arabinoside (AraC; 300 microM) induced a time-dependent accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase-3. Moreover, Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-fmk), a CPP32-selective antagonist, dose-dependently suppressed AraC-induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil-DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC-treated cells. The more alkaline isoforms, 1-3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4-6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h; a 4-h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside-induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC-induced neuronal death.     

Noninvasive delivery of small inhibitory RNA and other reagents to pulmonary alveoli in mice

A technically easy, noninvasive means of delivering molecules to alveoli, which act selectively or specifically in the lung, would be experimentally and therapeutically useful. As proof of principle, we took advantage of the spreading ability of pulmonary surface active material (InfaSurf), mixed it with elastase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) small inhibitory RNA (siRNA), or all-trans retinoic acid (ATRA), and instilled microliter amounts of the mixture into the nose of lightly anesthetized mice. One instillation of elastase caused diffuse alveolar destruction (emphysema) demonstrating widespread alveolar delivery. A single nasal instillation of GAPDH siRNA, compared with scrambled GAPDH siRNA, lowered GAPDH protein in lung, heart, and kidney by approximately 50-70% 1 and 7 days later. To test the possibility of lung-specific delivery of a potentially therapeutic drug, we administered ATRA and monitored its effect on expression of cellular retinol binding protein (CRBP)-1 mRNA, whose translation product is a key molecule in retinoid metabolism. Given intranasally, ATRA elevated CRBP-1 mRNA 4.3-fold in a lung-specific manner. The same dose and dose schedule of ATRA given intraperitoneally increased CRBP-1 mRNA only approximately 1.8-fold in lung; intraperitoneally administered ATRA elevated expression of CRBP-1 mRNA 1.7-fold or more in brain cortex, cerebellum, and testes, thereby increasing the risk of untoward effects. This simple noninvasive technique allows regulation of specific proteins in the lung and lung-specific delivery of reagents of experimental and potentially therapeutic importance.     

Binding of estrogen and progesterone-BSA conjugates to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the effects of the free steroids on GAPDH enzyme activity: physiological implications

In this study rat brain solubilized plasmalemma-microsomal fractions (B-P3) or cytosolic fractions were applied to P-3-BSA (progesterone linked to BSA at C-3 position) and E-6-BSA (17beta-estradiol linked to BSA at C-6 position) affinity columns. It is interesting that a 37 kDa protein was retained by both columns which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal sequencing. The 37 kDa protein (GAPDH) was not retained by either a control BSA conjugated affinity column or a corticosterone-BSA affinity column. E-6-BSA bound to GAPDH with higher binding affinity than P-3-BSA or T-3-BSA (testosterone linked to BSA at C-3 position) affinity columns. In addition, the binding of 17beta-E-6-BSA to GAPDH was impeded by free estrogen (17beta-estradiol) completely. Binding studies of E-6-BSA and P-3-BSA to commercial GAPDH from rabbit skeletal muscle using radiolabeled ligand binding assays revealed that P-3-BSA had 10x lower GAPDH binding affinity than E-6-BSA. Next, the effects of estrogen and progesterone on GAPDH activity were studied. Rapid and significant increases in V(max) and changes in K(m) were observed by the addition of 10 nM estradiol, whereas 100 nM progesterone decreased only V(max) significantly. Testosterone, corticosterone, 17alpha-estradiol, and diethylstilbestrol did not affect the enzyme activity. The results indicate that GAPDH is a target site for 17beta-estradiol and progesterone and suggest possible roles in the regulation of cellular metabolism and synaptic remodeling in which GAPDH has been reported to be involved.     

Disclosure of a pro-apoptotic glyceraldehyde-3-phosphate dehydrogenase promoter: anti-dementia drugs depress its activation in apoptosis

Overexpression and subsequent nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in neuronal apoptosis induced by several stimuli in which GAPDH antisense oligonucleotides specifically block the increment (2 approximately 3 fold) of GAPDH mRNA contents occurring prior to neuronal death. However, these agents do not affect the basal, constitutive mRNA contents. This suggests that there may be distinct gene regulations for GAPDH mRNA expression. Herein, we cloned two types of promoter regions upstream of this gene; viz., #104 (1.02-kb) and #302 (2.46-kb). These fragments were inserted into the pGL3 luciferase reporter system and transiently transfected into cultured cerebellar neurons undergoing cytosine arabinonucleoside-induced apoptosis. The functional analysis of these constructs revealed that #104, but not #302, increased luciferase activity in response to the apoptotic stimulus. Deletion and replacement mutation analysis of the #104 fragment disclosed the promoter core harbored between the 154-bp and 84-bp domains (3.5-fold activity of the control). Furthermore, anti-dementia drugs (such as Cognex and Aricept) markedly depress the expression of this pro-apoptotic GAPDH promoter activity. Interestingly, immunocytochemical examination of human post-mortem materials from patients with Alzheimer's disease revealed nuclear aggregated GAPDH in neurons of the affected brain regions, implying an association with apoptotic cell death. The current findings indicate that induction of the pro-apoptotic protein GAPDH is genetically regulated at the level of promoter activation, and this protein may be an important molecular target for developing anti-apoptotic therapeutic agents in certain neurological illnesses.     

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.     

Purification and identification of an IgE suppressor from strawberry in an in vitro immunization system

We purified and identified an IgE suppressor from the strawberry 'Toyonoka', based on the decrease of IgE production in in vitro immunization (IVI). Gel filtration experiment indicated that fractions in a 15-48 kDa range and <10 kDa have an IgE suppressive activity. Furthermore, the fraction in 15-48 kDa was subjected to chromatofocusing and found to have activities at isoelectric points, pI 6.0, 7.0, and 8.0-9.2. We focused on the active fractions of pI 8.0-9.2 and the purified a large amount of strawberry extracts by cation exchange resins in batch. A purified 39 kDa protein showed homology to plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in N-terminal amino acid sequence and had GAPDH enzymatic activity. Nucleotide sequence and deduced amino acid sequence of the obtained cDNA clone of the protein matched with the sequence of Fragaria x ananassa GAPDH in the GenBank with >98% identical nucleotides and >99% identical amino acids, respectively. The purified strawberry GAPDH suppressed total IgE production in IVI in a dose-dependent manner. From these results, we identified GAPDH as IgE suppressor in the strawberry. Our study may be applicable to the development of new methods to relieve allergic conditions using GAPDH and the screening of other functional factors for human health.     

The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer

Although glyceraldehyde-3-phosphate dehydrogenase's (GAPDH) predilection for AU-rich elements has long been known, the expected connection between GAPDH and control of mRNA stability has never been made. Recently, we described GAPDH binding the AU-rich terminal 144 nt of the colony-stimulating factor-1 (CSF-1) 3' untranslated region (UTR), which we showed to be an mRNA decay element in ovarian cancer cells. CSF-1 is strongly correlated with the poor prognosis of patients with ovarian cancer. We investigated the functional significance of GAPDH's association with CSF-1 mRNA and found that GAPDH small interfering RNA reduces both CSF-1 mRNA and protein levels by destabilizing CSF-1 mRNA. CSF-1 mRNA half-lives were decreased by 50% in the presence of GAPDH small interfering RNA. RNA footprinting analysis of the 144 nt CSF-1 sequence revealed that GAPDH associates with a large AU-rich-containing region. The effects of binding of GAPDH protein or ovarian extracts to mutations of the AU-rich regions within the footprint were consistent with this finding. In a tissue array containing 256 ovarian and fallopian tube cancer specimens, we found that GAPDH was regulated in these cancers, with almost 50% of specimens having no GAPDH staining. Furthermore, we found that low GAPDH staining was associated with a low CSF-1 score (P = 0.008). In summary, GAPDH, a multifunctional protein, now adds regulation of mRNA stability to its repertoire. We are the first to evaluate the clinical role of GAPDH protein in cancer. In ovarian cancers, we show that GAPDH expression is regulated, and we now recognize that one of the many functions of GAPDH is to promote mRNA stability of CSF-1, an important cytokine in tumor progression.     

Dual transgenic reporter mice as a tool for monitoring expression of glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein of astrocytes, and its expression changes dramatically during development and following injury. To facilitate study of the regulation of GFAP expression, we have generated dual transgenic mice expressing both firefly luciferase under the control of a 2.2 kb human GFAP promoter and Renilla luciferase under the control of a 0.5 kb human Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) promoter for normalization of the GFAP signal. The GFAP-fLuc was highly expressed in brain compared to other tissues, and was limited to astrocytes, whereas the GAPDH-RLuc was more widely expressed. Normalization of the GFAP signal to the GAPDH signal reduced the inter-individual variability compared to using the GFAP signal alone. The GFAP/GAPDH ratio correctly reflected the up-regulation of GFAP that occurs following retinal degeneration in FVB/N mice because of the rd mutation. Following kainic acid-induced seizures, changes in the GFAP/GAPDH ratio precede those in total GFAP protein. In knock-in mice expressing the R236H Alexander disease mutant, GFAP promoter activity is only transiently elevated and may not entirely account for the accumulation of GFAP protein that takes place.     

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.     

美洲大蠊3-磷酸甘油醛脱氢酶基因的克隆及表达

旨在通过5’-RACE获得美洲大蠊3-磷酸甘油醛脱氢酶(GAPDH)基因的全长cDNA序列,进行生物信息学分析,构建原核表达载体,诱导重组蛋白表达,为进一步研究其功能奠定基础.通过3’-RACE技术,PCR扩增获取编码美洲大蠊GAPDH蛋白的全长cDNA序列;采用生物信息学方法推导出该序列编码的氨基酸序列及其理化性质;预测信号肽、蛋白疏水性、可溶性、跨膜区结构、二级结构、三级结构,并构建系统发育树;构建原核表达载体pET28a-GAPDH,IPTG诱导重组蛋白表达,并用Histag抗体Western blotting验证.结果显示,美洲大蠊GAPDH基因,其完整阅读框含999个碱基,编码332个氨基酸.序列分析显示,该蛋白与家蚕GAPDH相似性为89％,具有GAPDH保守功能域,经IPTG诱导获得重组蛋白.