Effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor,OdhI, involved in glutamate production in Corynebacterium glutamicum

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (ODH) complex is negatively regulated by the unphosphorylated form of OdhI protein, which is critical for L-glutamate overproduction. We examined the potential impact of protein acylation at lysine (K)-132 of OdhI in C. glutamicum ATCC13032. The K132E succinylation-mimic mutation reduced the ability of OdhI to bind OdhA, the catalytic subunit of the ODH complex, which reduced the inhibition of ODH activity. In vitro succinylation of OdhI protein also reduced the ability to inhibit ODH, and the K132R mutation blocked the effect. These results suggest that succinylation at K132 may attenuate the OdhI function. Consistent with these results, the C. glutamicum mutant strain with OdhI-K132E showed decreased L-glutamate production. Our results indicated that not only phosphorylation but also succinylation of OdhI protein may regulate L-glutamate production in C. glutamicum.     

Crystal structure of the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex

The thiamine-dependent E1o component (EC 1.2.4.2) of the 2-oxoglutarate dehydrogenase complex catalyses a rate-limiting step of the tricarboxylic acid cycle (TCA) of aerobically respiring organisms. We describe the crystal structure of Escherichia coli E1o in its apo and holo forms at 2.6 A and 3.5 A resolution, respectively. The structures reveal the characteristic fold that binds thiamine diphosphate and resemble closely the alpha(2)beta(2) hetero-tetrameric E1 components of other 2-oxo acid dehydrogenase complexes, except that in E1o, the alpha and beta subunits are fused as a single polypeptide. The extended segment that links the alpha-like and beta-like domains forms a pocket occupied by AMP, which is recognised specifically. Also distinctive to E1o are N-terminal extensions to the core fold, and which may mediate interactions with other components of the 2-oxoglutarate dehydrogenase multienzyme complex. The active site pocket contains a group of three histidine residues and one serine that appear to confer substrate specificity and the capacity to accommodate the TCA metabolite oxaloacetate. Oxaloacetate inhibits E1o activity at physiological concentrations, and we suggest that the inhibition may allow coordinated activity within the TCA cycle. We discuss the implications for metabolic control in facultative anaerobes, and for energy homeostasis of the mammalian brain.     

MRPS36 provides a structural link in the eukaryotic 2-oxoglutarate dehydrogenase complex

The tricarboxylic acid cycle is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled from three catalytically active subunits (E1o, E2o, E3). In fungi and animals, however, the protein MRPS36 has more recently been proposed as a putative additional component. Based on extensive cross-linking mass spectrometry data supported by phylogenetic analyses, we provide evidence that MRPS36 is an important member of the eukaryotic OGDHC, with no prokaryotic orthologues. Comparative sequence analysis and computational structure predictions reveal that, in contrast with bacteria and archaea, eukaryotic E2o does not contain the peripheral subunit-binding domain (PSBD), for which we propose that MRPS36 evolved as an E3 adaptor protein, functionally replacing the PSBD. We further provide a refined structural model of the complete eukaryotic OGDHC of approximately 3.45 MDa with novel mechanistic insights.     

Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet C NMR of the adult mouse brain

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the ¹³C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-¹³C₂]glucose (oxidized by both neurons and glia) and [1,2-¹³C₂]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by ¹³C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of ¹³C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The ¹³C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-¹³C]acetate relative to [1,6-¹³C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.     

Glutamate production by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>: dependence on the oxoglutarate dehydrogenase inhibitor protein OdhI and protein kinase PknG

We recently showed that the activity of the 2-oxoglutarate dehydrogenase complex (ODHC) in Corynebacterium glutamicum is controlled by a novel regulatory mechanism that involves a 15-kDa protein called OdhI and serine/threonine protein kinase G (PknG). In its unphosphorylated state, OdhI binds to the E1 subunit (OdhA) of ODHC and, thereby, inhibits its activity. Inhibition is relieved by phosphorylation of OdhI at threonine-14 by PknG under conditions requiring high ODHC activity. In this work, evidence is provided that the dephosphorylation of phosphorylated OdhI is catalyzed by a phospho-Ser/Thr protein phosphatase encoded by the gene cg0062, designated ppp. As a decreased ODHC activity is important for glutamate synthesis, we investigated the role of OdhI and PknG for glutamate production under biotin limitation and after addition of Tween-40, penicillin, or ethambutol. A ΔodhI mutant formed only 1–13% of the glutamate synthesized by the wild type. Thus, OdhI is essential for efficient glutamate production. The effect of a pknG deletion on glutamate synthesis was dependent on the induction conditions. Under strong biotin limitation and in the presence of ethambutol, the ΔpknG mutant showed significantly increased glutamate production, offering a new way to improve production strains. Dedicated to Prof. Dr. Hermann Sahm on the occasion of his 65th birthday     

Rre37 stimulates accumulation of 2-oxoglutarate and glycogen under nitrogen starvation in Synechocystis sp. PCC 6803

Rre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Δrre37 was due to low expression of glycogen anabolic genes. In addition to low 2-oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose-6-phosphate, fructose-1,6-bisphosphate, and glyceraldehyde-3-phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2-oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation.     

Brain pyruvate and 2-oxoglutarate dehydrogenase complexes are mitochondrial targets of the CoA ester of the Refsum disease marker phytanic acid

Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC(50) approximately 10(-6)-10(-7) M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refsum disease.     

Catalytic domain of Salmonella typhimurium 2-oxoglutarate dehydrogenase is localized in N-terminal region

2-Oxoglutarate dehydrogenase (lipoamide) [OGDH or E1o: 2-oxoglutarate: lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinating); EC 1.2.4.2] is a component enzyme of the 2-oxoglutarate dehydrogenase complex. Salmonella typhimurium gene encoding OGDH (ogdh) has been cloned in Escherichia coli. The libraries were screened for the expression of OGDH by complementing the gene in E. coli E1o-deficient mutant. Three positive clones (named Odh-3, Odh-5 and Odh-7) contained the identical 2.9 kb Sau3AI fragment as determined by restriction mapping and Southern hybridization, and expressed OGDH efficiently and constitutively using its own promoter in the heterologous host. This gene spans 2878 bases and contains an open reading frame of 2802 nucleotides encoding a mature protein of 927 amino acid residues (M_r=110,000). The comparison of the deduced amino acid sequence of the cloned OGDH with E. coli OGDH shows 91% sequence identity. To localize the catalytic domain responsible for E. coli E1o-complementation, several deletion mutants lacking each portion of the ogdh gene were constructed using restriction enzymes. From the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a polypeptide which showed a complementation activity with an M_r of 30,000 was detected. The catalytic domain was localized in N-terminal region of the gene. Therefore, this is a first identification of the catalytic domain in bacterial ogdh gene.     

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.     

Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of Neurospora crassa and compared localization with the pyruvate dehydrogenase complex

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.     

Involvement of Ca/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus

Although multifunctional Ca²⁺/calmodulin-dependent protein kinases (CaM-kinases) are widely distributed in animal cells, the occurrence of CaM-kinases in the basidiomycetous mushroom has not previously been documented. When the extracts from various developmental stages from mycelia to the mature fruiting body of Coprinus cinereus were analyzed by Western blotting using Multi-PK antibodies, which had been generated to detect a wide variety of protein serine/threonine kinases (Ser/Thr kinases), a variety of stage-specific Ser/Thr kinases was detected. Calmodulin (CaM) overlay assay using digoxigenin-labeled CaM detected protein bands of 65 kDa, 58 kDa, 46 kDa, 42 kDa, and 38 kDa only in the presence of CaCl₂, suggesting that these bands were CaM-binding proteins. When the CaM-binding fraction was prepared from mycelial extract of C. cinereus by CaM-Sepharose and analyzed with Multi-PK antibodies, two major immunoreactive bands corresponding to 65 kDa and 46 kDa were detected. CaM-binding fraction, thus obtained, exhibited Ca²⁺/CaM-dependent protein kinase activity toward protein substrates such as histones. These CaM-kinases were found to be highly expressed in the actively growing mycelia, but not in the resting mycelial cells. Mycelial growth was enhanced by the addition of CaCl₂ in the culture media, but inhibited by the addition of EGTA or trifluoperazine, a potent CaM inhibitor. This suggested that CaM-dependent enzymes including CaM-kinases play crucial roles in mycelial growth of basidiomycete C. cinereus.     

Evidence for functional and regulatory cross-talk between the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex and 2-oxoadipate dehydrogenase on the l-lysine,l-hydroxylysine and l-tryptophan degradation pathways from studies in vitro

Herein are reported findings in vitro suggesting both functional and regulatory cross-talk between the human 2-oxoglutarate dehydrogenase complex (hOGDHc), a key regulatory enzyme within the tricarboxylic acid cycle (TCA cycle), and a novel 2-oxoadipate dehydrogenase complex (hOADHc) from the final degradation pathway of l-lysine, l-hydroxylysine and l-tryptophan. The following could be concluded from our studies by using hOGDHc and hOADHc assembled from their individually expressed components in vitro: (i) Different substrate preferences (k_cat/K_m) were displayed by the two complexes even though they share the same dihydrolipoyl succinyltransferase (hE2o) and dihydrolipoyl dehydrogenase (hE3) components; (ii) Different binding modes were in evidence for the binary hE1o-hE2o and hE1a-hE2o subcomplexes according to fluorescence titrations using site-specifically labeled hE2o-derived proteins; (iii) Similarly to hE1o, the hE1a also forms the ThDP-enamine radical from 2-oxoadipate (electron paramagnetic resonance detection) in the oxidative half reaction; (iv) Both complexes produced superoxide/H₂O₂ from O₂ in the reductive half reaction suggesting that hE1o, and hE1a (within their complexes) could both be sources of reactive oxygen species generation in mitochondria from 2-oxoglutarate and 2-oxoadipate, respectively; (v) Based on our findings, we speculate that hE2o can serve as a trans-glutarylase, in addition to being a trans-succinylase, a role suggested by others; (vi) The glutaryl-CoA produced by hOADHc inhibits hE1o, as does succinyl-CoA, suggesting a regulatory cross-talk between the two complexes on the different metabolic pathways.     

Crystal structure of an asymmetric complex of pyruvate dehydrogenase kinase 3 with lipoyl domain 2 and its biological implications

A homodimer of pyruvate dehydrogenase kinase (PDHK) is an integral part of pyruvate dehydrogenase complex (PDC) to which it is anchored primarily through the inner lipoyl-bearing domains (L2) of transacetylase component. The catalytic cycle of PDHK and its translocation over the PDC surface is thought to be mediated by the "symmetric" and "asymmetric" modes, in which the PDHK dimer binds to two and one L2-domain(s), respectively. Whereas the structure of the symmetric PDHK/L2 complex was reported, the structural organization and functional role of the asymmetric complex remain obscure. Here, we report the crystal structure of the asymmetric PDHK3/L2 complex that reveals several functionally important features absent from the previous structures. First, the PDHK3 subunits have distinct conformations: one subunit exhibits "open" and the other "closed" configuration of the putative substrate-binding cleft. Second, access to the closed cleft is additionally restricted by local unwinding of the adjacent alpha-helix. Modeling indicates that the target peptide might gain access to the PDHK active center through the open but not through the closed cleft. Third, the ATP-binding loop in one PDHK3 subunit adopts an open conformation, implying that the nucleotide loading into the active site is mediated by the inactive "pre-insertion" binding mode. Altogether our data suggest that the asymmetric complex represents a physiological state in which binding of a single L2-domain activates one of the PDHK protomers while inactivating another. Thus, the L2-domains likely act not only as the structural anchors but also modulate the catalytic cycle of PDHK.     

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury,preventing inactivation of the 2-oxoglutarate dehydrogenase complex

Background and purpose

Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine.

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK₁ and glnK₂. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK₁ and GlnK₂. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK₁ and GlnK₂ produced equivalent results.     

Partially unfolded forms and non-two-state folding of a beta-sandwich: FHA domain from Arabidopsis receptor kinase-associated protein phosphatase

FHA domains adopt a beta-sandwich fold with 11 strands. The first evidence of partially unfolded forms of a beta-sandwich is derived from native-state hydrogen exchange (NHX) of the forkhead-associated (FHA) domain from kinase-associated protein phosphatase from Arabidopsis. The folding kinetics of this FHA domain indicate that EX2 behavior prevails at pH 6.3. In the chevron plot, rollover in the folding arm and bends in the unfolding arm suggest folding intermediates. NHX of this FHA domain suggests a core of six most stable beta-strands and two loops, characterized by rare global unfolding events. Flanking this stable core are beta-strands and recognition loops with less stability, termed subglobal motifs. These suggest partially unfolded forms (near-native intermediates) with two levels of stability. The spatial separation of the subglobal motifs on the flanks suggests possible parallelism in their folding as additional beta-strands align with the stable core of six strands. Intermediates may contribute to differences in stabilities and m-values suggested by NHX or kinetics relative to chemical denaturation. Residual structure in the unfolded regime is suggested by superprotection of beta-strand 6 and by GdmCl-dependence of adjustments in amide NMR spectra and residual optical signal. The global folding stability depends strongly on pH, with at least 3 kcal/mol more stability at pH 7.3 than at pH 6.3. This FHA domain is hypothesized to fold progressively with initial hydrophobic collapse of its stable six-stranded core followed by addition of less stable flanking beta-strands and ordering of recognition loops.     

The human GLUD2 glutamate dehydrogenase and its regulation in health and disease

Whereas glutamate dehydrogenase in most mammals (hGDH1 in the human) is encoded by a single functional GLUD1 gene expressed widely, humans and other primates have acquired through retroposition an X-linked GLUD2 gene that encodes a highly homologous isoenzyme (hGDH2) expressed in testis and brain. Using an antibody specific for hGDH2, we showed that hGDH2 is expressed in testicular Sertoli cells and in cerebral cortical astrocytes. Although hGDH1 and hGDH2 have similar catalytic properties, they differ markedly in their regulatory profile. While hGDH1 is potently inhibited by GTP and may be controlled by the need of the cell for ATP, hGDH2 has dissociated its function from GTP and may metabolize glutamate even when the Krebs cycle generates GTP amounts sufficient to inactivate hGDH1. As astrocytes are known to provide neurons with lactate that largely derives from the Krebs cycle via conversion of glutamate to α-ketoglutarate, the selective expression of hGDH2 may facilitate metabolic recycling processes essential for glutamatergic transmission. As there is evidence for deregulation of glutamate metabolism in degenerative neurologic disorders, we sequenced GLUD1 and GLUD2 genes in neurologic patients and found that a rare T1492G variation in GLUD2 that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 interacted significantly with Parkinson's disease (PD) onset. Thus, in two independent Greek and one North American PD cohorts, Ser445Ala hemizygous males, but not heterozygous females, developed PD 6-13 years earlier than subjects with other genotypes. The Ala445-hGDH2 variant shows enhanced catalytic activity that is resistant to modulation by GTP, but sensitive to inhibition by estrogens. These observations are thought to suggest that enhanced glutamate oxidation by the Ala445-hGDH2 variant accelerates nigral cell degeneration in hemizygous males and that inhibition of the overactive enzyme by estrogens protects heterozygous females. We then evaluated the interaction of estrogens and neuroleptic agents (haloperidol and perphenazine) with the wild-type hGDH1 and hGDH2 and found that both inhibited hGDH2 more potently than hGDH1 and that the evolutionary Arg443Ser substitution was largely responsible for this sensitivity. Hence, the properties acquired by hGDH2 during its evolution have made the enzyme a selective target for neuroactive steroids and drugs, providing new means for therapeutic interventions in disorders linked to deregulation of this enzyme.     

Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications

Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.8 A resolution. The structure reveals that PDP1 belongs to the PPM family of protein serine/threonine phosphatases, which, in spite of a low level of sequence identity, share the structural core consisting of the central beta-sandwich flanked on both sides by loops and alpha-helices. Consistent with the previous studies, two well-fixed magnesium ions are coordinated by five active site residues and five water molecules in the PDP1c catalytic center. Structural analysis indicates that, while the central portion of the PDP1c molecule is highly conserved among the members of the PPM protein family, a number of structural insertions and deletions located at the periphery of PDP1c likely define its functional specificity towards the PDC. One notable feature of PDP1c is a long insertion (residues 98-151) forming a unique hydrophobic pocket on the surface that likely accommodates the lipoyl moiety of the E2 domain in a fashion similar to that of PDHKs. The cavity, however, appears more open than in PDHK, suggesting that its closure may be required to achieve tight, specific binding of the lipoic acid. We propose a mechanism in which the closure of the lipoic acid binding site is triggered by the formation of the intermolecular (PDP1c/L2) Ca2+ binding site in a manner reminiscent of the Ca2+ -induced closure of the regulatory domain of troponin C.     

The two catalytic components of the 2-oxoglutarate dehydrogenase complex in rat cerebral synaptic and nonsynaptic mitochondria: Comparison of the response to in vitro treatment with ammonia,hyperammonemia, and hepatic encephalopathy

The effects of in vitro treatment with ammonium chloride, hepatic encephalopathy (HE) due to thioacetamide (TAA) induced liver failure and chronic hyperammonemia produced by i.p. administration of ammonium acetate on the two components of the multienzyme 2-oxoglutarate dehydrogenase complex (OGDH): 2-oxoglutarate decarboxylase (E1) and lipoamide dehydrogenase (E3), were examined in synaptic and nonsynaptic mitochondria from rat brain. With regard to E1 the response to ammonium ions in vitro (3 mM NH₄Cl) was observed in nonsynaptic mitochondria only and was manifested by a 21% decrease of Vmax and a 35% decrease of Km for 2-oxoglutarate (2-OG). By contrast, both in vivo conditions primarily affected the synaptic mitochondrial E1: TAA-induced HE produced an 84% increase of Vmax and a 38% increase of Km for 2-OG. Hyperammonemia elevated Vmax of E1 by 110% and Km for 2-OG by 30%. HE produced no effect at all in nonsynaptic mitochondria while hyperammonemia produced a 35% increase of Vmax and a 30% increase of Km for 2-OG of E1. Both in vivo conditions produced a 20% increase of E3 activity in synaptic mitochondria, but no effect at all in nonsynaptic mitochondria. The preferential sensitivity of E1 to ammonium chloride in vitro in nonsynaptic mitochondria and hyperammonemic conditions in vivo in synaptic mitochondria may play a crucial role in the compartmentation of OGDH responses under analogous conditions. These results confirm the intrinsic differences between the OGDH properties in the synaptic and nonsynaptic brain compartments.     

Proliferating cell nuclear antigen in the cytoplasm interacts with components of glycolysis and cancer

Proliferating cell nuclear antigen (PCNA) is involved in a wide range of functions in the nucleus. However, a substantial amount of PCNA is also present in the cytoplasm, although their function is unknown. Here we show, through Far-Western blotting and mass spectrometry, that PCNA is associated with several cytoplasmic oncoproteins, including elongation factor, malate dehydrogenase, and peptidyl-prolyl isomerase. Surprisingly, PCNA is also associated with six glycolytic enzymes that are involved in the regulation of steps 4-9 in the glycolysis pathway.

Structured summary

MINT-7995351: G3P (uniprotkb:P04406) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescencemicroscopy (MI:0416)MINT-7995334: ENOA (uniprotkb:P06733) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescencemicroscopy (MI:0416)MINT-7995368: ALDOA (uniprotkb:P04075) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescencemicroscopy (MI:0416)MINT-7995141: G3P (uniprotkb:P04406) binds (MI:0407) to PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995182: ENOA (uniprotkb:P06733) binds (MI:0407) to PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995132: G3P (uniprotkb:P04406) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995228: PRDX6 (uniprotkb:P30041) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995220: CAH2 (uniprotkb:P00918) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995114: Triosephosphateisomerase (uniprotkb:P60174) binds (MI:0407) to PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995244: K2C7 (uniprotkb:P08729) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995252: ANXA2 (uniprotkb:P07355) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995122: Triosephosphateisomerase (uniprotkb:P60174) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995093: ALDOA (uniprotkb:P04075) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995148: PGK1 (uniprotkb:P00558) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995158: PGAM1 (uniprotkb:P18669) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995166: PGAM1 (uniprotkb:P18669) binds (MI:0407) to PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995105: ALDOA (uniprotkb:P04075) binds (MI:0407) to PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995260: PPIA (uniprotkb:P62937) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995173: ENOA (uniprotkb:P06733) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995268: EF1A (uniprotkb:P68104) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995236: MDHM (uniprotkb:P40926) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995189: RSSA (uniprotkb:P08865) physicallyinteracts (MI:0915) with PCNA (uniprotkb:P12004) by farwesternblotting (MI:0047)MINT-7995282: PCNA (uniprotkb:P12004) physicallyinteracts (MI:0915) with ALDOA (uniprotkb:P00883) and G3P (uniprotkb:P46406) by antibaitcoimmunoprecipitation (MI:0006).