Cytosolic Malate Dehydrogenase Research Articles

FLOURY ENDOSPERM16 encoding a NAD-dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice.

SummaryStarch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map‐based cloning and complementation tests demonstrated that FLO16 encodes a NAD‐dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis‐related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.

Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from -1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme's thermal responses and foster evolutionary adaptation of function.

Protein profile of Dabry’s sturgeon (Acipenser dabryanus) spermatozoa and relationship to sperm quality

Knowledge of conditions affecting sperm quality is essential for efficient culture of fish for commercial purposes and conservation of species. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry were used to characterize the proteomic profile of Acipenser dabryanus spermatozoa relative to motility and fertilization capacity. There were differential amounts of protein in 313 spots in spermatozoa of males classified to have relatively greater or lesser spermatozoa quality. The functions of 43 of 50 selected proteins were identified. The proteins in 14 spots were involved in metabolism, and of these, proteins in 11 spots were highly abundant in spermatozoa of males categorized to have spermatozoa of greater quality, including pyruvate kinase, enolase B, phosphoglycerate kinase, lactate dehydrogenase, cytosolic malate dehydrogenase, brain creatine kinase b, Ckmb protein, and nucleoside diphosphate kinase. The proteins involved in mechanics of flagellum movement were identified, including the dynein intermediate chain, radial spoke head 1 homolog; ropporin-1-like, Bardet-Biedl syndrome 5, ADP-ribosylation factor-like protein 3, tektin-4, gamma-actin, and tubulin cytoskeleton proteins to be differentially abundant in spermatozoa that were classified relatively greater or lesser quality. Heat shock proteins, copper/zinc superoxide dismutase and peroxiredoxins, which are involved in stress response were of differential abundance in spermatozoa from males with spermatozoa in the two different classification groups. Proteins were also detected that are involved in protein folding and binding, or hydrolase activity. The results are valuable for the prediction of sperm quality and for reproduction management in A. dabryanus and other threatened species.

Structure of glyoxysomal malate dehydrogenase (MDH3) from Saccharomyces cerevisiae.

Malate dehydrogenase (MDH), a carbohydrate and energy metabolism enzyme in eukaryotes, catalyzes the interconversion of malate to oxaloacetate (OAA) in conjunction with that of nicotinamide adenine dinucleotide (NAD+) to NADH. Three isozymes of MDH have been reported in Saccharomyces cerevisiae: MDH1, MDH2 and MDH3. MDH1 is a mitochondrial enzyme and a member of the tricarboxylic acid cycle, whereas MDH2 is a cytosolic enzyme that functions in the glyoxylate cycle. MDH3 is a glyoxysomal enzyme that is involved in the reoxidation of NADH, which is produced during fatty-acid β-oxidation. The affinity of MDH3 for OAA is lower than those of MDH1 and MDH2. Here, the crystal structures of yeast apo MDH3, the MDH3-NAD+ complex and the MDH3-NAD+-OAA ternary complex were determined. The structure of the ternary complex suggests that the active-site loop is in the open conformation, differing from the closed conformations in mitochondrial and cytosolic malate dehydrogenases.

Characterization of the Genes Involved in Malic Acid Metabolism from Pear Fruit and Their Expression Profile after Postharvest 1-MCP/Ethrel Treatment.

In this study, five genes involved in malic acid (MA) metabolism, including a cytosolic NAD-dependent malate dehydrogenase gene ( cyNAD-MDH), a cytosolic NADP-dependent malic enzyme gene ( cyNADP-ME), two vacuolar H+-ATPase genes ( vVAtp1 and vVAtp2), and one vacuolar inorganic pyrophosphatase gene ( vVPp), were characterized from pear fruit based on bioinformatic and experimental analysis. Their expression profile in "Housui" pear was tissue-specific, and their expression patterns during fruit development were diverse. During "Housui" pear storage, MA content decreased, which was associated with the downregulated transcripts of MA metabolism-related genes and cyNAD-MDH activity and higher cyNADP-ME activity. The response of MA metabolism to postharvest 1.5 μL L-1 1-MCP fumigation and 0.5 mL L-1 ethrel dipping was distinct: 1-MCP fumigation upregulated gene expression and cyNAD-MDH activity and suppressed cyNADP-ME activity, and thus maintained higher MA abundance when compared with those in the control; on the other hand, an opposite behavior was observed in ethrel-treated fruit.

Malate valves: old shuttles with new perspectives.

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyse the reversible interconversion of malate and oxaloacetate and their transport. Depending on the co‐enzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes. Activities of NAD‐dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids. In addition, chloroplasts possess a NADP‐dependent MDH isoform. The NADP‐MDH as part of the ‘light malate valve’ plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post‐translational redox‐modification mediated via the ferredoxin‐thioredoxin system and fine control via the NADP +/NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD‐MDH (‘dark malate valve’) is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, knowledge about regulation of the other two cytosolic MDHs as well as NAD‐MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria and peroxisomes have been characterised, but not much is known about cytosolic NAD‐MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange, focusing on the various metabolic functions of these valves.

A(xoneme) to z(inc): New paradigm of mammalian sperm zinc ion fluctuation from spermiogenesis to fertilization and its importance to livestock production

Knowledge of conditions affecting sperm quality is essential for efficient culture of fish for commercial purposes and conservation of species. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry were used to characterize the proteomic profile of Acipenser dabryanus spermatozoa relative to motility and fertilization capacity. There were differential amounts of protein in 313 spots in spermatozoa of males classified to have relatively greater or lesser spermatozoa quality. The functions of 43 of 50 selected proteins were identified. The proteins in 14 spots were involved in metabolism, and of these, proteins in 11 spots were highly abundant in spermatozoa of males categorized to have spermatozoa of greater quality, including pyruvate kinase, enolase B, phosphoglycerate kinase, lactate dehydrogenase, cytosolic malate dehydrogenase, brain creatine kinase b, Ckmb protein, and nucleoside diphosphate kinase. The proteins involved in mechanics of flagellum movement were identified, including the dynein intermediate chain, radial spoke head 1 homolog; ropporin-1-like, Bardet-Biedl syndrome 5, ADP-ribosylation factor-like protein 3, tektin-4, gamma-actin, and tubulin cytoskeleton proteins to be differentially abundant in spermatozoa that were classified relatively greater or lesser quality. Heat shock proteins, copper/zinc superoxide dismutase and peroxiredoxins, which are involved in stress response were of differential abundance in spermatozoa from males with spermatozoa in the two different classification groups. Proteins were also detected that are involved in protein folding and binding, or hydrolase activity. The results are valuable for the prediction of sperm quality and for reproduction management in A. dabryanus and other threatened species.

Redox signaling in plants

Hydrogen peroxide (H2O2) was once considered to be an unavoidable and toxic byproduct of an aerobic lifestyle. However, it is now recognized that endogenous H2O2 plays an important positive role as redox metabolite in cell signaling and it allows cells to rapidly respond to metabolic changes. These H2O2 signaling reactions need to be tightly controlled, and cells have developed elegant adaptive strategies to maintain the cellular redox state in balance to preventing damage. Focusing on structure and function of plant proteins, I will guide you in the world of thiol redox control and oxidative stress survival. – Waszczak, C. et al. Sulfenome mining in Arabidopsis thaliana. Proc Natl Acad Sci U S A 111, 11545-11550 (2014). – Akter, S. et al. DYn-2 Based Identification of Arabidopsis Sulfenomes. Mol Cell Proteomics 14, 1183-1200 (2015). – Bodra, N. et al. Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis. Sci Rep 7, 42494 (2017). – Huang, J. et al. Self-protection of cytosolic malate dehydrogenase against oxidative stress in Arabidopsis. J Exp Bot (2017). – Tossounian, M. A. et al. Disulfide bond formation protects Arabidopsis thaliana glutathione transferase tau 23 from oxidative damage. Biochim Biophys Acta 1862, 775-789 (2018).

Creation of a formate: malate oxidoreductase by fusion of dehydrogenase enzymes with PEGylated cofactor swing arms

Enzymatic biocatalysis can be limited by the necessity of soluble cofactors. Here, we introduced PEGylated nicotinamide adenine dinucleotide (NAD(H)) swing arms to two covalently fused dehydrogenase enzymes to eliminate their nicotinamide cofactor requirements. A formate dehydrogenase and cytosolic malate dehydrogenase were connected via SpyCatcher-SpyTag fusions. Bifunctionalized polyethylene glycol chains tethered NAD(H) to the fusion protein. This produced a formate:malate oxidoreductase that exhibited cofactor-independent ping-pong kinetics with predictable Michaelis constants. Kinetic modeling was used to explore the effective cofactor concentrations available for electron transfer in the complexes. This approach could be used to create additional cofactor-independent transhydrogenase biocatalysts by swapping fused dehydrogenases.

A Biophysical Analysis of Malate Dehydrogenase and Citrate Synthase Protein‐Protein Interaction

The metabolon theory describes the protein interactions of enzymes regulating a metabolic pathway. Through these interactions proteins can share substrates through direct channeling. One set of enzymes that are involved in metabolon complexes are citrate synthase and malate dehydrogenase. This study attempts to understand the interactions regulating the metabolon of malate dehydrogenase (MDH) and citrate synthase (CS), which are integral intermediate metabolic enzymes. Using size exclusion chromatography and sedimentation, previous studies identified the interaction between CS and MDH to be isoform specific, but no further work has been conducted to obtain affinity values or the biophysical nature of the interaction. Codon optimized His tagged recombinant proteins were expressed and purified using affinity chromatography. These enzymes include: glyoxysomal malate dehydrogenase (gMDH), human cytosolic malate dehydrogenase (MDH1), human mitochondrial malate dehydrogenase (MDH2), and human mitochondrial citrate synthase (CS). We analyzed the protein interactions using fast thermal melts which further explain the thermal stability of the the dimerization of the metabolons. We first show that protein concentration did not induce oligomerization of CS or any MDH isoform, indicating that any changes are not from MDH or CS homodimerization. Next, we investigated specific CS‐MDH interactions using fast thermal melts. The results show that from the initial melting temperature of CS, the addition of mitochondrial MDH had a greater shift in its melting temperature than the addition of cytosolic MDH, by 3–4°C. The melting point of CS was not altered in the presence of the plant MDH isoform. This indicates a specific and dramatic set of contacts for mitochondrial MDH and CS. However unlike the information found in the literature there was still a measurable shift in melting point when cytosolic MDH was incubated with CS. Overall this shows for the first time a quantifiable interaction between proteins in a well‐established pathway that will allow the further dissection of these interactions.Support or Funding InformationSupport for this project was from NSF REU 146045This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and characterization of Taenia solium enolase as a plasminogen-binding protein

The larval stage of Taenia solium (cysticerci) is the causal agent of human and swine cysticercosis. When ingested by the host, T. solium eggs are activated and hatch in the intestine, releasing oncospheres that migrate to various tissues and evolve into cysticerci. Plasminogen (Plg) receptor proteins have been reported to play a role in migration processes for several pathogens. This work is aimed to identify Plg-binding proteins in T. solium cysticerci and determine whether T. solium recombinant enolase (rTsEnoA) is capable of specifically binding and activating human Plg. To identify Plg-binding proteins, a 2D-SDS-PAGE ligand blotting was performed, and recognized spots were identified by MS/MS. Seven proteins from T. solium cysticerci were found capable of binding Plg: fascicilin-1, fasciclin-2, enolase, MAPK, annexin, actin, and cytosolic malate dehydrogenase. To determine whether rTsEnoA binds human Plg, a ligand blotting was performed and the results were confirmed by ELISA both in the presence and absence of εACA, a competitive Plg inhibitor. Finally, rTsEnoA-bound Plg was activated to plasmin in the presence of tPA. To better understand the evolution of enolase isoforms in T. solium, a phylogenetic inference analysis including 75 enolase amino acid sequences was conducted. The origin of flatworm enolase isoforms, except for Eno4, is independent of their vertebrate counterparts. Therefore, herein we propose to designate tapeworm protein isoforms as A, B, C, and 4. In conclusion, recombinant enolase showed a strong plasminogen binding and activating activity in vitro. T. solium enolase could play a role in parasite invasion along with other plasminogen-binding proteins.

Early quantitative profiling of differential retinal protein expression in lens-induced myopia in guinea pig using fluorescence difference two-dimensional gel electrophoresis.

The current study aimed to investigate the differential protein expression in guinea pig retinas in response to lens-induced myopia (LIM) before fully compensated eye growth. Four days old guinea pigs (n=5) were subjected to −4D LIM for 8 days. Refractive errors were measured before and at the end of the lens wear period. Ocular dimensions were also recorded using high-frequency A-scan ultrasonography. After the LIM treatment, retinas of both eyes were harvested and soluble proteins were extracted. Paired retinal protein expressions in each animal were profiled and compared using a sensitive fluorescence difference two-dimensional gel electrophoresis. The quantitative retinal proteomes of myopic and control eye were analysed using computerised DeCyder software. Those proteins that were consistently changed with at least 1.2-fold difference (P<0.05) in the same direction in all five animals were extracted, trypsin digested and identified by tandem mass spectrometry. Significant myopia was induced in guinea pigs after 8 days of lens wear. The vitreous chamber depth in lens-treated eyes was found to be significantly elongated. Typically, more than 1,000 protein spots could be detected from each retina. Thirty-two of them showed differential expression between myopic and untreated retina. Among these proteins, 21 spots were upregulated and 11 were downregulated. Eight protein spots could be successfully identified which included β-actin, enolase 1, cytosolic malate dehydrogenase, Ras-related protein Rab-11B, protein-L-isoaspartate (D-aspartate) O-methyltransferase, PKM2 protein, X-linked eukaryotic translation initiation factor 1A and ACP1 protein. The present study serves as the first report to uncover the retinal 2D proteome expressions in mammalian guinea pig myopia model using a top-down fluorescent dyes labelling gel approach. The results showed a downregulation in glycolytic enzymes that may suggest a significant alteration of glycolysis during myopia development. Other protein candidates also suggested multiple pathways which could provide new insights for further study of the myopic eye growth.

Structural flexibility and protein adaptation to temperature: Molecular dynamics analysis of malate dehydrogenases of marine molluscs

Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural "flexibility." However, quantifying flexibility and comparing flexibility across proteins has remained a challenge. To address this issue, we examined temperature effects on cytosolic malate dehydrogenase (cMDH) orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of ∼60 °C. We describe consistent patterns of convergent evolution in adaptation of function [temperature effects on KM of cofactor (NADH)] and structural stability (rate of heat denaturation of activity). To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis, we performed molecular dynamics simulation (MDS) analyses. MDS analyses revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements [root mean square deviation (rmsd) of main-chain atoms]. Root mean square fluctuations (RMSFs) of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs. Regions of sequence involved in ligand binding and catalysis-termed mobile regions 1 and 2 (MR1 and MR2), respectively-showed the largest values for RMSF. Heat-induced changes in RMSF values across the sequence and, importantly, in MR1 and MR2 were greatest in cold-adapted species. MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.

Self-protection of cytosolic malate dehydrogenase against oxidative stress in Arabidopsis.

Plant malate dehydrogenase (MDH) isoforms are found in different cell compartments and function in key metabolic pathways. It is well known that the chloroplastic NADP-dependent MDH activities are strictly redox regulated and controlled by light. However, redox dependence of other NAD-dependent MDH isoforms have been less studied. Here, we show by in vitro biochemical characterization that the major cytosolic MDH isoform (cytMDH1) is sensitive to H2O2 through sulfur oxidation of cysteines and methionines. CytMDH1 oxidation affects the kinetics, secondary structure, and thermodynamic stability of cytMDH1. Moreover, MS analyses and comparison of crystal structures between the reduced and H2O2-treated cytMDH1 further show that thioredoxin-reversible homodimerization of cytMDH1 through Cys330 disulfide formation protects the protein from overoxidation. Consistently, we found that cytosolic thioredoxins interact specifically with cytMDH in a yeast two-hybrid system. Importantly, we also show that cytosolic and chloroplastic, but not mitochondrial NAD-MDH activities are sensitive to H2O2 stress in Arabidopsis. NAD-MDH activities decreased both in a catalase2 mutant and in an NADP-thioredoxin reductase mutant, emphasizing the importance of the thioredoxin-reducing system to protect MDH from oxidation in vivo. We propose that the redox switch of the MDH activity contributes to adapt the cell metabolism to environmental constraints.

Metabolism of Citrate and Other Carboxylic Acids in Erythrocytes As a Function of Oxygen Saturation and Refrigerated Storage.

State-of-the-art proteomics technologies have recently helped to elucidate the unanticipated complexity of red blood cell metabolism. One recent example is citrate metabolism, which is catalyzed by cytosolic isoforms of Krebs cycle enzymes that are present and active in mature erythrocytes and was determined using quantitative metabolic flux analysis. In previous studies, we reported significant increases in glycolytic fluxes in red blood cells exposed to hypoxia in vitro or in vivo, an observation relevant to transfusion medicine owing to the potential benefits associated with hypoxic storage of packed red blood cells. Here, using a combination of steady state and quantitative tracing metabolomics experiments with 13C1,2,3-glucose, 13C6-citrate, 13C515N2-glutamine, and 13C1-aspartate via ultra-high performance liquid chromatography coupled on line with mass spectrometry, we observed that hypoxia in vivo and in vitro promotes consumption of citrate and other carboxylates. These metabolic reactions are theoretically explained by the activity of cytosolic malate dehydrogenase 1 and isocitrate dehydrogenase 1 (abundantly represented in the red blood cell proteome), though moonlighting functions of additional enzymes cannot be ruled out. These observations enhance understanding of red blood cell metabolic responses to hypoxia, which could be relevant to understand systemic physiological and pathological responses to high altitude, ischemia, hemorrhage, sepsis, pulmonary hypertension, or hemoglobinopathies. Results from this study will also inform the design and testing of novel additive solutions that optimize red blood cell storage under oxygen-controlled conditions.

The Proteome Response of \u201cHordeum marinum\u201d to Long-term Salinity Stress

Salinity is a major constraint to crop productivity and mechanisms of plant responses to salinity stress are extremely complex. “Hordeum marinum” is a salt tolerant barley species, which could be a good source to evaluate salt-tolerance patterns. Proteomics is a powerful technique to identify proteins involved in plant adaptation to stresses. We applied a proteomic approach to better understanding the mechanism of plant responses to salinity in a salt-tolerant genotype of barley. At the 4-leaf stage, plants were exposed to 0 (control treatment) or 300 mM NaCl (salt treatment). Salt treatment was maintained for 3 weeks. Total proteins of leaf 4 were extracted and separated by two-dimensional gel electrophoresis. More than 290 protein spots were reproducibly detected. Of these, 20 spots showed significant changes to salt treatment compared to the control: 19 spots were upregulated and 1 spot was absent. Using MALDI-TOF/TOF MS, we identified 20 cellular proteins which represented 11 different proteins and were classified into five categories. These proteins were involved in various cellular functions. Upregulation of proteins which involved in protein processing (ribosomal protein, cullin family, cp31AHv protein and RNA recognition motif (RRM) superfamily), photosynthesis (Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and Ribulose bisphosphate carboxylase/oxygenase activase (rubisco activase)), energy metabolism (cytosolic malate dehydrogenase (cyMDH) and fructokinase), oxygen species scavenging and defense (cystatin and thioredoxin) may increase plant adaptation to salt stress.

Heat-resistant cytosolic malate dehydrogenases (cMDHs) of thermophilic intertidal snails (genus Echinolittorina): protein underpinnings of tolerance to body temperatures reaching 55°C

Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41-44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the Michaelis-Menten constant of the cofactor NADH, KMNADH) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners (E. malaccana and E. radiata) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata, in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases (positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on fine-scale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation.

Snails prioritise protein stability at sizzling temperatures

No matter how warm it gets outside, most endotherms maintain a reasonably stable internal temperature; if they don't, there's usually a very good reason for their temperature to rocket. However, the body temperatures of ectothermic species that cling to life on the rocky tidal shore are not so dependable. When the tide is out and some molluscs are left high and dry on the sea shore, their body temperatures can soar to levels where the delicate protein structures within cells could begin to unravel. Yet, the remarkable Echinolittorina family of periwinkle molluscs routinely experience perilously high body temperatures in excess of 50°C as they bake in the sun. Intrigued by the extraordinary resilience of these diminutive creatures, George Somero from Stanford University, USA, and Yun-wei Dong from Xiamen University, China, decided to find out how members of the robust family reinforce delicate protein structures so that they remain intact at temperatures where other proteins would disintegrate.Choosing the ubiquitous cytosolic enzyme malate dehydrogenase, which produces malate for use by mitochondria in the production of ATP, Ming-ling Liao and Shu Zhang collected two members of the Echinolittorina family – E. malaccana, which can tolerate temperatures in excess of 55°C, and the less robust E. radiata – to find out what gives E. malaccana proteins the thermal edge. After isolating the enzyme from both molluscs, Liao and S. Zhang provided the enzymes with oxaloacetic acid at temperatures ranging from 20 to 40°C and measured the rate of conversion of the acid to malate. They found that E. malaccana malate dehydrogenase functioned better at high temperatures than the E. radiata enzyme. And when the duo measured the proteins’ stability by holding them at temperatures up to 57.5°C and recording how their activity altered over an hour, the E. radiata malate dehydrogenase was completely inactive by the end of the experiment, whereas the more resilient E. malaccana protein was still functional, although at a much lower rate.Curious to find out why the E. malaccana enzyme was so much tougher than the E. radiata malate dehydrogenase, Liao and S. Zhang analysed the sequence of amino acids in both proteins and identified two key locations – position 48 and 114 in the peptide chain – where the serine amino acids in the E. radiata protein were replaced by glycine amino acids, which are much smaller, in E. malaccana malate dehydrogenase. Wondering how these subtle differences might affect the proteins, Liao, Guang-ya Zhang and Yun-meng Chu calculated how increasing heat would affect the proteins’ function by effectively boiling them up to 42 and 57°C in computer simulations that replicated the effects of different temperatures on the delicate protein structures. Analysing the calculations, the team found that the core of the E. malaccana enzyme was much more stable at 57°C than that of the E. radiata enzyme. However, the simulations also showed that regions of the more heat-resistant protein that were involved in the conversion of oxaloacetic acid into malate became more flexible. The team suggests that these local increases in flexibility could permit the enzyme to continue functioning at lower temperatures while allowing the protein to remain stable at higher temperatures where other proteins would collapse.

Cytosolic malate dehydrogenase activity helps support glycolysis in actively proliferating cells and cancer.

Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD and therapies designed at targeting glycolysis must consider both dehydrogenases.

Molecular characterization of a cytosolic malate dehydrogenase gene(GhcMDH1) from cotton

Malate dehydrogenase(MDH) is a key enzyme that catalyzes the reversible oxidation of oxaloacetate to malate and plays an important role in the physiological processes of plant growth and development. However, cyto-solic malate dehydrogenase(cMDH), which is crucial for malate synthesis in the cytosol, still has not been extensively characterized in plants. Here, we isolated a cytosolic malate dehydrogenase gene, designated as GhcMDH1, from Gossypium hirsutum and characterized its possible molecular function in cotton fiber. The cloned cDNA of GhcMDH1 is 1520 base pairs in length, and has an open reading frame of 999 base pairs, encoding for 332 amino acid residues with an estimated molecular weight of 35580 and pI of 6.35. Sequence alignment showed that the de-duced amino acid sequence of GhcMDH1 protein shared a high similarity to other plant cMDHs. Confocal and im-munological analysis confirmed that GhcMDH1 protein was subcellularly localized to the cytosol. Quantitative real-time polymerase chain reaction(PCR) revealed that GhcMDH1 was constitutively expressed in all vegetative cotton tissues, with slightly lower levels in roots than stems and leaves. Interestingly, the transcripts of GhcMDH1 were detected in 5―25 d post anthesis(DPA) fibers and highly abundant at 15 DPA fibers. The total MDH activities and malate contents of cotton fibers were positively correlated with the fiber elongation rates, suggesting that GhcMDH1 may function in malate synthesis in fast fiber elongation. In agreement with this suspicion, the recombi-nant His-GhcMDH1 protein mainly drives the reaction towards malate generation in vitro. In conclusion, our mole-cular characterization of the GhcMDH1 gene provides valuable insights to further investigate the malate equilibrium in cotton fiber development.