succinyl-CoA Synthetase Subunit Research Articles

Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel invivo model of mitochondrial myopathy. The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model invivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, exvivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in exvivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.

Systemic alterations of tricarboxylic acid cycle enzymes in Alzheimer's disease.

Mitochondrial dysfunction, especially tricarboxylic acid (TCA) cycle arrest, is strongly associated with Alzheimer's disease (AD), however, its systemic alterations in the central and peripheral of AD patients are not well defined. Here, we performed an integrated analysis of AD brain and peripheral blood cells transcriptomics to reveal the expression levels of nine TCA cycle enzymes involving 35 genes. The results showed that TCA cycle related genes were consistently down-regulated in the AD brain, whereas 11 genes were increased and 16 genes were decreased in the peripheral system. Pearson analysis of the TCA cycle genes with Aβ, Tau and mini-mental state examination (MMSE) revealed several significant correlated genes, including pyruvate dehydrogenase complex subunit (PDHB), isocitrate dehydrogenase subunits (IDH3B, IDH3G), 2-oxoglutarate dehydrogenase complex subunit (DLD), succinyl-CoA synthetase subunit (SUCLA2), malate dehydrogenase subunit (MDH1). In addition, SUCLA2, MDH1, and PDHB were also uniformly down-regulated in peripheral blood cells, suggesting that they may be candidate biomarkers for the early diagnosis of AD. Taken together, TCA cycle enzymes were systemically altered in AD progression, PDHB, SUCLA2, and MDH1 may be potential diagnostic and therapeutic targets.

Succinyl-CoA Synthetase Dysfunction as a Mechanism of Mitochondrial Encephalomyopathy: More than Just an Oxidative Energy Deficit.

Biallelic pathogenic variants in subunits of succinyl-CoA synthetase (SCS), a tricarboxylic acid (TCA) cycle enzyme, are associated with mitochondrial encephalomyopathy in humans. SCS catalyzes the interconversion of succinyl-CoA to succinate, coupled to substrate-level phosphorylation of either ADP or GDP, within the TCA cycle. SCS-deficient encephalomyopathy typically presents in infancy and early childhood, with many patients succumbing to the disease during childhood. Common symptoms include abnormal brain MRI, basal ganglia lesions and cerebral atrophy, severe hypotonia, dystonia, progressive psychomotor regression, and growth deficits. Although subunits of SCS were first identified as causal genes for progressive metabolic encephalomyopathy in the early 2000s, recent investigations are now beginning to unravel the pathomechanisms underlying this metabolic disorder. This article reviews the current understanding of SCS function within and outside the TCA cycle as it relates to the complex and multifactorial mechanisms underlying SCS-related mitochondrial encephalomyopathy.

Value of Immunohistochemical Expression of Apelin, Succinate Dehydrogenase B, Chromogranin B, Human Epidermal Growth Factor Receptor-2, Contactin 4, and Succinyl-CoA Synthetase Subunit Beta in Differentiating Metastatic From Non-Metastatic Pheochromocytoma and Paraganglioma.

ObjectiveWe aimed to retrospectively collect pathologically identified pheochromocytoma and paraganglioma (PPGL) tumor tissues from our center and investigate the expression of apelin and succinyl-CoA synthetase subunit beta (SUCLG2), human epidermal growth factor receptor-2 (HER2 or ERBB-2), contactin 4 (CNTN4), chromogranin B (CHGB), and succinate dehydrogenase B (SDHB) in metastatic and non-metastatic PPGLs, for exploring their roles in the diagnosis of metastatic PPGLs.MethodsA total of 369 patients with pathologically and surgically confirmed PPGLs at Xiangya Hospital, Central South University, between June 2010 and June 2020 were retrospectively included. Sixty patients—12 patients with metastatic PPGLs and 48 patients with non-metastatic PPGLs—were selected through propensity score matching (1:4) to reduce the effect of PPGL type, sex, and age. We observed and quantified the expression of apelin, SDHB, CHGB, ERBB-2, CNTN4, and SUCLG2 in paraffin-embedded samples using immunohistochemical staining.ResultsNo significant differences were observed between the metastatic group and non-metastatic group with respect to the expression of CNTN4 and SUCLG2. The expression of apelin, SDHB, CHGB, and ERBB-2 was significantly different between the two groups. The expression of apelin, SDHB, and CHGB was significantly lower in the metastatic group than that in the non-metastatic group (P < 0.001). ERBB-2 expression was significantly higher in the metastatic group than in the non-metastatic group (P = 0.042). Kaplan–Meier analysis revealed that patients with negative expression of apelin, SDHB, and CHGB showed significantly lower metastasis-free survival than those with positive expression. Multivariate Cox analysis revealed that SDHB and CHGB levels were independently associated with metastasis-free survival.ConclusionThe expression levels of apelin, CHGB, SDHB, and ERBB-2 may be predictive biomarkers for the diagnosis of metastatic PPGLs. Patients with negative expression of apelin, CHGB, and SDHB should be subjected to frequent postoperative follow-up procedures

The Role of TCA Cycle Compounds in Erythroid Heme Synthesis and Its Regulation During an Inflammatory Response

One early hallmark event of terminal erythroid differentiation is the induction of heme and globin synthesis. Onset of heme synthesis occurs when the first pathway enzyme, erythroid-specific 5-aminolevulinate synthase (ALAS2), is induced. The steps of heme biosynthesis are identical between non-erythroid (housekeeping) and differentiating erythroid cells, but two genes encode the first enzyme, 5-aminolevulinate synthase (ALAS). The housekeeping gene (ALAS1) is expressed in non-erythroid cells while the erythroid-specific gene (ALAS2) is expressed only in the erythron. Heme synthesis in metazoan cells requires iron, glycine, and succinyl CoA. The supply of iron required for heme synthesis has been well studied and a reasonable view of the components involved in this process exists. Glycine is abundant in plasma (~250 µM) and studies of glycine transporters are consistent with glycine being supplied from extracellular sources. For succinyl CoA the general assumption is that it is produced by the TCA cycle. This may be possible for housekeeping heme synthesis where only modest levels of heme are made at a given time, but this mechanism would clearly place cells under strain during erythroid differentiation when cells make ~109 molecules of heme in a span of 2-3 days.ALAS2, but not ALAS1, physically interacts with the succinyl CoA synthetase (SCS) ATP-dependent subunit SUCLA2. Since ALAS utilizes succinyl CoA to synthesize aminolevulinic acid (ALA), and because SUCLA2 as a subunit of SCS may be involved in the ATP-dependent reverse reaction of SCS to generate succinyl CoA from succinate, one popular hypothesis is that the ALAS2-SUCLA2 interaction exists to provide succinyl CoA for ALAS2. While the evidence for an association between ALAS2 and SUCLA2 is strong, there is no experimental data showing that SUCLA2 has an impact on ALAS2 activity or that there is a role for this interaction in the supply of succinyl CoA for ALAS2.In the current work we have sought to determine if succinyl CoA for heme synthesis during erythropoiesis in MEL cells originates from the TCA cycle or more directly from metabolism of glutamine via transaminases and α-ketoglutarate dehydrogenase (α-KGDH). Employing 13C-labeled glucose, diethyl succinate, or glutamine we determined that succinate is a poor source of carbons for erythroid heme synthesis, providing a maximum of 5 carbons out of a possible 26 (by isotopomer analysis) and labeling only 2.5% of carbons of isolated heme. Glucose contributes up to 18 carbons and a total of 8.3%, and glutamine contributes to 23 carbons and a total of 23%. Enzyme assays of TCA cycle enzymes revealed that during erythropoiesis only aconitase, isocitrate dehydrogenase, and α-KGDH are induced, which is consistent with glutamine, and not succinate, being the major source of carbon for heme.We also examined the impact of itaconate, a compound produced from the TCA cycle in LPS-induced macrophages, on erythroid differentiation. Itaconate is known to inhibit succinate dehydrogenase, and so might be expected to induce heme synthesis if succinyl CoA is being supplied from accumulating succinate via ATP-dependent SCS. However, dimethyl itaconate (a cell permeable form of itaconate) inhibits heme synthesis in differentiating erythroid K562 cells and this inhibition is reversed by ALA. It has long been assumed that SCS has broad specificity and is capable of making itaconyl CoA (Adler J. et al. 1957, JBC 229:865). However, we found that purified human and porcine SCS do not synthesize itaconyl CoA, but that the enzyme succinyl CoA:glutarate CoA transferase (SUGCT) generates itaconyl CoA from itaconate and succinyl CoA. Additionally we demonstrated that itaconyl CoA inhibits purified ALAS2 at µM concentrations. Previously published data along with our studies on K562 cells in the presence of LPS-induced RAW cells suggest that during the inflammatory response macrophages resident in erythroblastic islands produce itaconate as well as cytokines that induce proximal developing red cells to express a plasmalemmal transporter (possibly SLC13A5) required for itaconate import. Intracellular itaconate is then converted to itaconyl CoA via SUGCT, resulting in ALAS2 inhibition and a concomitant decrease in heme synthesis. This process restricts the amount of protoporphyrin synthesized during infection and, thereby, complements mechanisms that restrict the availability of iron to microbial pathogens. DisclosuresNo relevant conflicts of interest to declare.

Glutamine via α-ketoglutarate dehydrogenase provides succinyl-CoA for heme synthesis during erythropoiesis

AbstractDuring erythroid differentiation, the erythron must remodel its protein constituents so that the mature red cell contains hemoglobin as the chief cytoplasmic protein component. For this, ∼109 molecules of heme must be synthesized, consuming 1010 molecules of succinyl-CoA. It has long been assumed that the source of succinyl-coenzyme A (CoA) for heme synthesis in all cell types is the tricarboxylic acid (TCA) cycle. Based upon the observation that 1 subunit of succinyl-CoA synthetase (SCS) physically interacts with the first enzyme of heme synthesis (5-aminolevulinate synthase 2, ALAS2) in erythroid cells, it has been posited that succinyl-CoA for ALA synthesis is provided by the adenosine triphosphate–dependent reverse SCS reaction. We have now demonstrated that this is not the manner by which developing erythroid cells provide succinyl-CoA for ALA synthesis. Instead, during late stages of erythropoiesis, cellular metabolism is remodeled so that glutamine is the precursor for ALA following deamination to α-ketoglutarate and conversion to succinyl-CoA by α-ketoglutarate dehydrogenase (KDH) without equilibration or passage through the TCA cycle. This may be facilitated by a direct interaction between ALAS2 and KDH. Succinate is not an effective precursor for heme, indicating that the SCS reverse reaction does not play a role in providing succinyl-CoA for heme synthesis. Inhibition of succinate dehydrogenase by itaconate, which has been shown in macrophages to dramatically increase the concentration of intracellular succinate, does not stimulate heme synthesis as might be anticipated, but actually inhibits hemoglobinization during late erythropoiesis.

Proteomics and 1H NMR-based metabolomics analysis of pathogenic Vibrio vulnificus aquacultures isolated from sewage drains.

Vibrio bacteria live in both marine and freshwater habitats and are associated with aquatic animals. Vibrio vulnificus is a pathogenic bacterium that infects people and livestock. It is usually found in offshore waters or within fish and shellfish. This study presents a comparative proteomic analysis of the outer membrane protein (OMP) changes in V. vulnificus proteins after stimulation with sewage from sewage drains. Using two-dimensional electrophoresis followed by MALDI-TOF MS/MS, 32 protein spots with significant differences in abundance were identified and characterized. These identified proteins were found to be involved in various functional categories, including catalysis, transport, membrane proteins progresses, receptor activity, energy metabolism, cytokine activity, and protein metabolism. The mRNA expression levels of 12 differential proteins were further assessed by qRT-PCR. Seven genes including carboxypeptidase, hemoglobin receptor, succinate dehydrogenase iron-sulfur subunit, ATP synthase subunit alpha, thioredoxin, succinyl-CoA synthetase subunit, and alanine dehydrogenase were downregulated upon stimulation, whereas the protein expression levels HupA receptor, type I secretion outer membrane protein, glutamine synthetase, superoxide dismutase, OmpU, and VuuA were upregulated. 1H NMR spectra showed 18 dysregulated metabolites from V. vulnificus after the sewage stimulation and the pathogenicity was enhanced after that.

Succinyl-CoA Synthetase: New Antigen Candidate of Bartonella bacilliformis.

BackgroundBartonella bacilliformis is the causative agent of Carrion’s disease, a neglected illness with mortality rates of 40–85% in the absence of treatment. The lack of a diagnostic technique to overcome misdiagnosis and treat asymptomatic carriers is of note. This study aimed to identify new B. bacilliformis antigenic candidates that could lead to a new diagnostic tool able to be implemented in endemic rural areas.Methodology/Principal FindingsBlood (n = 198) and serum (n = 177) samples were collected in northern Peru. Clinical data were recorded. Specific 16S rRNA amplification by RT-PCR, IFA and ELISA for IgM/IgG with whole cells as antigens was done. Western blot analysis and N-terminal amino acid sequencing detected seroreactive proteins. ELISAs for IgM/IgG for the antigenic candidates were performed. Of the population 33.3% reported at least one symptom compatible with Carrion’s disease; 25.4% (IFA), 27.1% (ELISA-IgG), 33.9% (ELISA-IgM) and 38.9% (RT-PCR) of samples were positive. Four proteins were considered potential antigenic candidates, including two new antigenic candidates, succinyl-CoA synthetase subunit α (SCS-α) and succinyl-CoA synthetase subunit β (SCS-β). On Western blot both Pap31 and SCS-α interacted with IgM, while GroEL and SCS-β interacted with IgG. The presence of specific antibodies against the antigenic candidates varied from 34.5% (IgG against SCS-α) to 97.2% (IgM against Pap31).Conclusions/SignificanceRT-PCR and the high levels of positivity for specific ELISAs demonstrate high levels of B. bacilliformis exposure and asymptomatic carriers among inhabitants. The new antigens identified might be used as a new rapid diagnostic tool to diagnose acute Carrion’s disease and identify asymptomatic carriers.

Differences between high- and low-motility buffalo sperm identified by comparative proteomics.

This study was undertaken to investigate differences in protein expression between high- and low-motility sperm of swamp buffalo. The research used two-dimensional gel electrophoresis (2DE) coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS) to analyse the different proteins. The results showed 18 different expression protein spots between high- and low-motility buffalo sperm; eight of these proteins were up-regulated in low-motility sperm, five were down-regulated, one deleted and four proteins specifically expressed. Finally, four proteins were successfully identified by MS as belonging to three unique proteins; they are outer dense fibre of sperm tails protein 2 (ODF2), ATP synthase subunit alpha (ATP5A1) and succinyl-CoA synthetase subunit beta (SUCLG2). In summary, these results help to develop an understanding of the molecular mechanisms associated with low-motility sperm and provide clues for finding molecular markers associated with sperm motility.

Cold shock CspA and CspB protein production during periodic temperature cycling in Escherichia coli

BackgroundTemperature is an important environmental factor which can dramatically affect biochemical processes in bacteria. Temperatures above optimal cause heat shock, while low temperatures induce cold shock. Since the physiological response of the bacterium Escherichia coli to slow temperature fluctuation is not well known, we investigated the effect of periodic temperature cycling between 37° and 8°C with a period of 2 h on proteome profile, cold shock CspA and CspB protein and gene production.ResultsSeveral proteins (i.e. succinyl-CoA synthetase subunit alpha, periplasmic oligopeptide-binding protein, maltose-binding periplasmic protein, outer membrane porin protein, flavodoxin-1, phosphoserine aminotransferase) were up or down regulated during temperature cycling, in addition to CspA and CspB production. The results indicate that transcription of cspA and cspB increased during each temperature downshift and consistently decreased after each temperature upshift. In sharp contrast CspA-FLAG and CspB-FLAG protein concentrations in the cell increased during the first temperature down-shift and remained unresponsive to further temperature fluctuations. The proteins CspA-FLAG and CspB-FLAG were not significantly degraded during the temperature cycling.ConclusionThe study demonstrated that slow periodic temperature cycling affected protein production compared to cells constantly incubated at 37°C or during classical cold shock. Bacterial cspA and cspB mRNA transcript levels fluctuated in synchrony with the temperature fluctuations. There was no corresponding pattern of CspA and CspB protein production during temperature cycling.

X-linked Sideroblastic Anemia Due to Carboxyl-terminal ALAS2 Mutations That Cause Loss of Binding to the β-Subunit of Succinyl-CoA Synthetase (SUCLA2)

Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the β subunit of succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for succinyl-CoA binding, an increased K(m) for succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.

Immunoproteomic Analysis of Chinese Brucella Vaccine Strain M5 and New Immunogenic Proteins

Brucellae are facultative intracellular gram-negative bacteria that cause human disease and significant worldwide economic loss due to infection of livestock. Available vaccines against Brucella spp. are live attenuated Brucella strains. In order to engineer a better vaccine to be used in animals and humans, our laboratory aims to develop an innocuous subunit vaccine. Particularly, we are interested in Brucella membrane proteins (MPs). In this study, an immunoproteomic approach was utilized to identify novel antigenic candidate proteins from Chinese Brucella vaccine strain M5 membrane proteins. The membrane proteins were separated by 2-DE and analyzed by western-blotting for their reactivity with the antiserum obtained from goats naturally infected by Brucella. Of a total of 9 immunogenic proteins identified, 7 were shown to be the novel antigens for Brucella spp. Some of the major protein components include outer-membrane protein OMP25, OMP31, and several new immunogenic proteins were identified, such as isovaleryl coenzyme A dehydrogenase, nitroglycerin reductase, succinyl-CoA synthetase subunit and S- adenosine -L- homocysteine hydrolase. Comparing these gene sequences revealed that 8 out of 9 immunoreactive protein genes were found in all 5 different Brucella strains. The elucidation of the immunome of Brucella vaccine strain M5 MPs identified a number of candidate proteins for developing vaccines against Brucella infection in livestock.

Differential Proteins of the Optic Ganglion in Octopus vulgaris Under Methanol Stress Revealed Using Proteomics

An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.

Fusion of the subunits α and β of succinyl-CoA synthetase as a phylogenetic marker for Pezizomycotina fungi

Gene fusions, yielding the formation of multidomain proteins, are evolutionary events that can be utilized as phylogenetic markers. Here we describe a fusion gene comprising the α and β subunits of succinyl-coA synthetase, an enzyme of the TCA cycle, in Pezizomycotina fungi. This fusion is present in all Pezizomycotina with complete genome sequences and absent from all other organisms. Phylogenetic analysis of the α and β subunits of succinyl-CoA synthetase suggests that both subunits were duplicated and retained in Pezizomycotina while one copy was lost from other fungi. One of the duplicated copies was then fused in Pezizomycotina. Our results suggest that the fusion of the α and β subunits of succinyl-CoA synthetase can be used as a molecular marker for membership in the Pezizomycotina subphylum. If a species has the fusion it can be reliably classified as Pezizomycotina, while the absence of the fusion is suggestive that the species is not a member of this subphylum.

A comparative study of the interaction of Bartonella henselae strains with human endothelial cells

Bartonella henselae can cause a wide range of clinical outcomes and may lead to severe disease, especially in patients with acquired immunodeficiency syndrome. It is well-known that B. henselae-induced cell proliferation is mediated by anti-apoptotic activity; however, the detailed mechanism is still unclear. In this study, the cellular responses of endothelial cells after infection with four B. henselae strains were compared and protein candidates that may be involved in the interaction between cells and bacteria were determined. The Houston-1 strain elicited the fastest response in terms of stimulating endothelial cell proliferation, and the JK-40 strain had the strongest ability to induce cell proliferation. By Western blot analysis, it was demonstrated that B. henselae-induced cell proliferation involved the mitochondria intrinsic apoptotic pathway. In addition, the adhesion abilities of the U-4 and JK-40 strains were much greater than those of the Houston-1 and JK-47 strains; however, the ability of Houston-1 to invade host cells was high. By two-dimensional gel electrophoresis analysis, it was found that succinyl-CoA synthetase subunit beta, phage-related protein, and ATP synthase subunit alpha might be involved in the invasion process. The expression of superoxide dismutase [Cu–Zn] precursor increased with infection time for all four strains but was significantly higher in the Houston-1 strain, which may increase the competitive advantage of Houston-1 in terms of survival in host cells and render it successful in invading host cells and stimulating cell proliferation. Our data suggest that the interaction of B. henselae and endothelial cells differed between strains, and the results indicated possible candidate proteins that may play a role in the pathogenesis of B. henselae infection.

Identification and Functional Analysis of Phosphoproteins Regulated by Auxin in Arabidopsis Roots

The phytohormone auxin modulates diverse aspects of plant growth and development. Protein phosphorylation is believed to play a key role in regulating auxin-mediated responses. To determine the phosphoproteins affected by auxin in Arabidopsis, we used phospho-specific antibodies to analyze their profiles on two-dimensional gels, then identified them by mass spectrometry. We found two phosphoproteins, enolase and the beta subunit of succinyl-CoA synthetase (SCS-beta), and noted that their phosphorylation was increased by auxin. To investigate their importance in auxin-mediated processes, we characterized Arabidopsis knockout mutants of the two genes. A homozygous null mutation in the gene for SCS-beta conferred embryo lethality. The enolase knockout mutants showed defects in root development similar to those of auxin-related mutants such as alf3 and xbat32. Therefore, we suggest that enolase is involved in auxin-regulated processes.

Identification and characterization of a novel protein histidine kinase in the islet β cell: evidence for its regulation by mastoparan, an activator of G-proteins and insulin secretion

Using insulin-secreting cells, we previously demonstrated that specific proteins associated with the cytosolic, secretory granule, and mitochondrial fractions undergo a novel type of phosphorylation on their histidine residues. Subsequently, we identified these proteins as the nucleoside diphosphate kinase (NDPK) [Kowluru and Metz, Biochemistry 1994;33:12495–503], the β subunit of trimeric GTP-binding proteins [Kowluru et al., Biochem J 1996;313:97–107], and the α subunit of succinyl-CoA synthetase [Kowluru, Diabetologia 2001;44:89–94], respectively. Since several other enzymes of intermediary metabolism (e.g. ATP-citrate lyase and glucose-6-phosphatase) also undergo histidine phosphorylation, these initial findings may have a more generalized significance to β cells. Herein, we characterized a novel protein histidine kinase in pancreatic β cells, and determined it to be acid- and heat-labile as well as alkali-resistant in its phosphorylation of histone 4. Such an activity was detected in normal rat islets, human islets, and clonal β (HIT-T15 and INS-1) cells, and could utilize either ATP or GTP as a phosphoryl donor (with K m values in the range of 60–100 μM). On a size-exclusion column, its molecular mass was estimated to be in the range of 60–70 kDa. It was stimulated by divalent cations (Mg 2+>Mn 2+>control=Ca 2+=Zn 2+=Co 2+), but was resistant to polyamines. It was inactivated by known in vitro inhibitors of protein histidine phosphorylation (e.g. UDP or cromoglycate). Mastoparan, a global activator of G-proteins and insulin secretion from isolated β cells, but not mastoparan-17, its inactive analog, stimulated histidine kinase activity and histidine phosphorylation of G β subunit and insulin secretion from isolated rat islets. These studies identify, for the first time, a protein kinase activity in the pancreatic β cell that does not act on traditional -Ser, -Tyr, or -Thr residues. They also establish a possible link between histidine kinase activity and G β phosphorylation in isolated β cells.

Adenine and guanine nucleotide-specific succinyl-CoA synthetases in the clonal beta-cell mitochondria: implications in the beta-cell high-energy phosphate metabolism in relation to physiological insulin secretion.

Succinyl-CoA synthetase catalyses the substrate level phosphorylation of ADP or GDP. It also supplies succinyl-CoA for heme synthesis. Recently, two distinct mitochondrial succinyl-CoA synthetase activities, one specific for ATP and the other for GTP, have been characterized in various tissues of pigeon. Because of the relative importance of mitochondrial high-energy phosphate metabolism in physiological insulin secretion and the few data available on mitochondrial succinyl-CoA synthetase in the beta cell, this study examined whether ATP-specific and GTP-specific succinyl-CoA synthetase activities are localized in the clonal beta-cell mitochondria. Using the mitochondrial extracts from clonal beta [INS-1 and HIT-T15] cells, we measured the formation of succinyl-CoA from succinate, CoA and ATP or GTP. To confirm the identity of these two enzymes, individual subunits of ATP-specific and GTP-specific to succinyl-CoA synthetase were identified by Western blot analysis. Both ATP-and GTP activities of succinylCoA synthetase were observed in the mitochondrial fractions from these cells. The ratios of GTP to ATP activities of succinyl-CoA synthetase were near unity in both of the cell types studied. Using affinity-purified antisera directed specifically against individual (alpha and beta) subunits of succinyl-CoA synthetase, we also identified both ATP-specific and GTP-specific forms of succinyl-CoA synthetase in HIT and INS cell mitochondria. Furthermore, using [gamma-32P]ATP as a phosphoryl donor, we observed that the alpha subunit of succinyl-CoA synthetase undergoes autophosphorylation at a histidine residue; co-provision of exogenous succinate and CoA resulted in pronounced dephosphorylation of the phosphorylated alpha subunit of succinyl-CoA synthetase. We provide evidence for the localization of two distinct activities of succinyl-CoA synthetase in the beta cell mitochondria. Whereas it is well established that ATP is critical for the beta cell mitochondrial metabolism, we propose that GTP generated by the activation of succinylCoA synthetase could promote key functional roles in the mitochondrial metabolism leading to insulin secretion.

Predicted structure and fold recognition for the glutamyl tRNA reductase family of proteins.

The conserved residues of glutamyl tRNA reductase (GTR) from Hordeum vulgare (GTRhorvu) were found from an alignment/pile-up of 24 homologous sequences found using BLAST searches. A multiple alignment of sequences was used to obtain a prediction of the secondary structure of the GTR's. This secondary structure was submitted to the THREADER program to find possible homologous 3D structures. To help select the template for predicting the fold for GTRhorvu, we employed both molecular-biological and biochemical information about GTRhorvu. After fitting the secondary structure of GTRhorvu to the selected template, the MODELLER program was used to determine the fold for GTRhorvu. This model was built using the B subunit of succinyl CoA synthetase, 1scuB, as a template for the 3D structure of GTRhorvu. From the predicted structure, possible regions were identified for the binding of glutamyl-tRNA, NADPH and a heme inhibitor. The predicted structure was used to propose a detailed biochemical mechanism for the GTR, involving Mg catalyzed thioester formation and reduction by NADPH to glutamate-1-semialdehyde. Sites for these reactions are identified. The predicted structure has been deposited in the Brookhaven database as ID 1b61.

Characterisation of several Hsp70 interacting proteins from mammalian organelles

Since both the spectrum and characteristics of in vivo substrates with affinity for Hsp70 members are largely unknown, we have investigated the range and type of mammalian organellar proteins which selectively interact with immobilised Escherichia coli Hsp70 (DnaK). Amongst a subset of organellar proteins selectively retained on DnaK, the major constituents represent unstable proteins and subunits of oligomeric proteins. The interactions with DnaK were diminished in the presence of mt-Hsp70 and BiP, while the complexes formed with DnaK were dissociated in the presence of K + and GrpE-like co-chaperones, suggesting that these organellar proteins constitute general Hsp70 substrates. Protein sequence analysis identified the major DnaK interacting constituents as the mitochondrial transcription factor A, the α- (but not the β-) subunit of succinyl CoA synthetase, mitochondrial 2,4-dienoyl CoA reductase, endoplasmic reticulum cyclophilin-B, peroxisomal multifunctional enzyme and a previously undescribed peroxisomal protein suspected to represent an isoform of 2,4-dienoyl CoA reductase. The selective retention of these fully synthesised proteins on Hsp70 most likely reflects the function of this molecular chaperone in protein biogenesis, but additionally, could extend the known functions of Hsp70 to include modulating the activities of certain proteins or enzymes which are important in cellular homeostasis.