Enzyme Pair Research Articles

Directed transport of neutrophil-derived extracellular vesicles enables platelet-mediated innate immune response.

The innate immune response to bacterial infections requires the interaction of neutrophils and platelets. Here, we show that a multistep reciprocal crosstalk exists between these two cell types, ultimately facilitating neutrophil influx into the lung to eliminate infections. Activated platelets adhere to intravascular neutrophils through P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1)-mediated binding, a primary interaction that allows platelets glycoprotein Ibα (GPIbα)-induced generation of neutrophil-derived extracellular vesicles (EV). EV production is directed by exocytosis and allows shuttling of arachidonic acid into platelets. EVs are then specifically internalized into platelets in a Mac1-dependent fashion, and relocated into intracellular compartments enriched in cyclooxygenase1 (Cox1), an enzyme processing arachidonic acid to synthesize thromboxane A2 (TxA2). Finally, platelet-derived-TxA2 elicits a full neutrophil response by inducing the endothelial expression of ICAM-1, intravascular crawling, and extravasation. We conclude that critical substrate–enzyme pairs are compartmentalized in neutrophils and platelets during steady state limiting non-specific inflammation, but bacterial infection triggers regulated EV shuttling resulting in robust inflammation and pathogen clearance.

Capturing a substrate in an activated RING E3/E2-SUMO complex.

SummaryPost-translational protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as small ubiquitin like modifier (SUMO) regulates processes including protein homeostasis, the DNA damage response, and the cell cycle. Proliferating cell nuclear antigen (PCNA) is modified by Ub or poly-Ub at Lys164 after DNA damage to recruit repair factors. Yeast PCNA is modified by SUMO on Lys164 and Lys127 during S-phase to recruit the anti-recombinogenic helicase Srs2. Lys164 modification requires specialized E2/E3 enzyme pairs for SUMO or Ub conjugation. For SUMO, Lys164 modification is strictly dependent on the E3 ligase Siz1, suggesting the E3 alters E2 specificity to promote Lys164 modification. The structural basis for substrate interactions in activated E3/E2-Ub/Ubl complexes remains unclear. Here, we report an engineered E2 protein and cross-linking strategies that trap an E3/E2-Ubl/substrate complex for structure determination, illustrating how an E3 can bypass E2 specificity to force-feed a substrate lysine into the E2 active site.

Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants.

BackgroundThe double digest restriction-site associated DNA sequencing technology (ddRAD-seq) is a reduced representation sequencing technology by sampling genome-wide enzyme loci developed on the basis of next-generation sequencing. ddRAD-seq has been widely applied to SNP marker development and genotyping on animals, especially on marine animals as the original ddRAD protocol is mainly built and trained based on animal data. However, wide application of ddRAD-seq technology in plant species has not been achieved so far. Here, we aim to develop an optimized ddRAD library preparation protocol be accessible to most angiosperm plant species without much startup pre-experiment and costs.ResultsWe first tested several combinations of enzymes by in silico analysis of 23 plant species covering 17 families of angiosperm and 1 family of bryophyta and found AvaII + MspI enzyme pair produced consistently higher number of fragments in a broad range of plant species. Then we removed two purifying and one quantifying steps of the original protocol, replaced expensive consumables and apparatuses by conventional experimental apparatuses. Besides, we shortened P1 adapter from 37 to 25 bp and designed a new barcode-adapter system containing 20 pairs of barcodes of varying length. This is an optimized ddRAD strategy for angiosperm plants that is economical, time-saving and requires little technical expertise or investment in laboratory equipment. We refer to this simplified protocol as MiddRAD and we demonstrated the utility and flexibility of our approach by resolving phylogenetic relationships of two genera of woody bamboos (Dendrocalamus and Phyllostachys). Overall our results provide empirical evidence for using this method on different model and non-model plants to produce consistent data.ConclusionsAs MiddRAD adopts an enzyme pair that works for a broad range of angiosperm plants, simplifies library constructing procedure and requires less DNA input, it will greatly facilitate designing a ddRAD project. Our optimization of this method may make ddRAD be widely used in fields of plant population genetics, phylogenetics, phylogeography and molecular breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0139-1) contains supplementary material, which is available to authorized users.

CD73 Inhibition Shifts Cardiac Macrophage Polarization toward a Microbicidal Phenotype and Ameliorates the Outcome of Experimental Chagas Cardiomyopathy.

Increasing evidence demonstrates that generation of extracellular adenosine from ATP, which is hydrolyzed by the CD39/CD73 enzyme pair, attenuates the inflammatory response and deactivates macrophage antimicrobial mechanisms. Although CD73 is emerging as a critical pathway and therapeutic target in cardiovascular disorders, the involvement of this ectonucleotidase during myocardial infection has not been explored. Using a murine model of infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy, we observed a sudden switch from the classical M1 macrophage (microbicidal) phenotype toward an alternative M2 (repairing/anti-inflammatory) phenotype that occurred within the myocardium very shortly after BALB/c mice infection. The observed shift in M1/M2 rate correlated with the cardiac cytokine milieu. Considering that parasite persistence within myocardium is a necessary and sufficient condition for the development of the chronic myocarditis, we hypothesized that CD73 activity may counteract cardiac macrophage microbicidal polarization, rendering the local immune response less effective. In fact, a transient treatment with a specific CD73 inhibitor (adenosine 5'-α,β-methylene-diphosphate) enhanced the microbicidal M1 subset predominance, diminished IL-4- and IL-10-producing CD4(+) T cells, promoted a proinflammatory cytokine milieu, and reduced parasite load within the myocardium during the acute phase. As a direct consequence of these events, there was a reduction in serum levels of creatine kinase muscle-brain isoenzyme, a myocardial-specific injury marker, and an improvement in the electrocardiographic characteristics during the chronic phase. Our results demonstrate that this purinergic system drives the myocardial immune response postinfection and harbors a promising potential as a therapeutic target.

Development of a universal double-digest RAD sequencing approach for a group of nonmodel, ecologically and economically important insect and fish taxa.

The generation of genome-scale data is critical for a wide range of questions in basic biology using model organisms, but also in questions of applied biology in nonmodel organisms (agriculture, natural resources, conservation and public health biology). Using a genome-scale approach on a diverse group of nonmodel organisms and with the goal of lowering costs of the method, we modified a multiplexed, high-throughput genomic scan technique utilizing two restriction enzymes. We analysed several pairs of restriction enzymes and completed double-digestion RAD sequencing libraries for nine different species and five genera of insects and fish. We found one particular enzyme pair produced consistently higher number of sequence-able fragments across all nine species. Building libraries off this enzyme pair, we found a range of usable SNPs between 4000 and 37000 SNPS per species and we found a greater number of usable SNPs using reference genomes than de novo pipelines in STACKS. We also found fewer reads in the Read 2 fragments from the paired-end Illumina Hiseq run. Overall, the results of this study provide empirical evidence of the utility of this method for producing consistent data for diverse nonmodel species and suggest specific considerations for sequencing analysis strategies.

Directional Regulation of Enzyme Pathways through the Control of Substrate Channeling on a DNA Origami Scaffold.

Artificial multi-enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH-MDH and G6pDH-LDH) through the control of NAD(+) substrate channeling by specifically shifting NAD(+) between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine.

Directional Regulation of Enzyme Pathways through the Control of Substrate Channeling on a DNA Origami Scaffold

AbstractArtificial multi‐enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH–MDH and G6pDH–LDH) through the control of NAD+ substrate channeling by specifically shifting NAD+ between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine.

Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity

Biomass deconstruction to small simple sugars is a potential approach to biofuels production; however, the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyperstable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. Proteins 2016; 84:1043-1054. © 2016 Wiley Periodicals, Inc.

Encapsulated Hydrogels by E-beam Lithography and Their Use in Enzyme Cascade Reactions.

Electron beam (e-beam) lithography was employed to prepare one protein immobilized hydrogel encapsulated inside another by first fabricating protein-reactive hydrogels of orthogonal reactivity and subsequently conjugating the biomolecules. Exposure of thin films of eight arm star poly(ethylene glycol) (PEG) functionalized with biotin (Biotin-PEG), alkyne (Alkyne-PEG) or aminooxy (AO-PEG) end-groups to e-beam radiation resulted in cross-linked hydrogels with the respective functionality. It was determined via confocal microscopy that a nominal size exclusion effect exists for streptavidin immobilized on Biotin-PEG hydrogels of feature sizes ranging from 5 to 40 μm. AO-PEG was subsequently patterned as an encapsulated core inside a contiguous outer shell of Biotin-PEG. Similarly, Alkyne-PEG was patterned as a core inside an AO-PEG shell. The hydrogel reactive end-groups were conjugated to dyes or proteins of complementary reactivity, and the three-dimensional (3-D) spatial orientation was determined for both configurations using confocal microscopy. The enzyme glucose oxidase (GOX) was immobilized in the core of the encapsulated Alkyne-PEG core/ AO-PEG shell architecture, and horseradish peroxidase (HRP) was conjugated to the shell periphery. Bioactivity for the HRP-GOX enzyme pair was observed in this encapsulated configuration by demonstrating that the enzyme pair was capable of enzyme cascade reactions.

Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes.

Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or pair of restriction enzymes on one sequenced genome, and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal, and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to eight times more genome coverage for the new combinations consisted of pairing four- or five-cutter restriction enzymes than the commonly used enzyme combination PstI + MspI. The empirical evaluation of the new enzyme combination (HinfI + HpyCH4IV) in 12 plant species showed 1.7–6 times more genome coverage than PstI + MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI + HpyCH4IV generated 7 and 1.3 times more SNPs (with 0–16.7% missing observations) than PstI + MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes.

Abstract B073: Structural basis of the 12-23 rule in V(D)J recombination

Abstract The V, D and J segment recombination (V(D)J recombination) is the key event in the development and maturation of T and B lymphocytes, which generates a diverse repertoire of T and B cell clones for the further selection of useful specificities. V(D)J recombination is also the basis for antibody diversity. The initiation step of V(D)J recombination is carried out by a pair of lymphocyte specific enzymes, which are called recombination-activating gene 1 and 2 (RAG1 and RAG2). RAG1 and RAG2 associate with each other and with two recognition signal sequences (RSS, 12RSS and 23RSS) adjacent to the coding ends of the V, D and J segments to catalyze the double stranded breaks required for joining. The reaction goes through several catalytic steps that result in formation of the single recombination complex (SC), the paired complex (PC), the cleaved signal complex (CSC) as well as the signal end complex (SEC). In order to understand the mechanisms of how RAG complex functions on DNA, we solved the structures PC and CSC by cryo-electron microscopy near the atomic resolution. Compared with the Apo-RAG complex that constitutes an open conformation, the PC and CSC reveal a closed conformation with RAG1 in one protomer interacts with RAG2 in another protomer. Furthermore, the first nucleotide of the heptamer in the non-transfer strand is flipped out to avoid the steric hindrance of the 3'-OH to attack the phosphodiester bond in the transfer strand. Upon nucleophilic attack and hairpin formation, the nucleotide adjacent to the 5'-position of the attacked phosphate is also flipped out to accommodate the tensile force in the hairpin DNA. In addition, we found that the conformation changes in the nonamer-binding domain (NBD), which is distorted toward to the 12RSS, and the distorsion requires two turns for the nonamer DNA in the 23RSS to reach the NBD. Finally, we found that two HMGB1 molecules, rather than one, are required to bind and bend both 12RSS and 23RSS DNA to adjust the conformational change of NBD on each side. In summary, our structural models not only uncover the mechanisms of how RAG protein catalyzes the hairpin formation, but also provide the structural basis of the 12-23 rule in V(D)J recombination. Citation Format: Heng Ru, Melissa G. Chambers, Maofu Liao, Hao Wu. Structural basis of the 12-23 rule in V(D)J recombination. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B073.

Analysis of DNA Methylation and Hydroxymethylation in the Genome of Crustacean Daphnia pulex.

The aim of our study was to analyze the presence of 5-methyl-cytosine (5-mC) and 5-hydroxymethyl-cytosine (5-hmC) in the genome of crustacean Daphnia pulex. First, the presence of 5-mC and 5-hmC in genomic DNA was demonstrated by using antibodies specific to either 5-mC or 5-hmC. Then, analysis of 5-mC and 5-hmC using pairs of restriction enzymes with different sensitivity to methylation and hydroxymethylation confirmed the presence of both modifications in selected regions of three genes (Cox4, Cand2 and Ephx1). To get a detailed picture of 5-hmC distribution over the D. pulex genome, we performed 5-hmC enrichment and sequenced the enriched fraction using next generation sequencing and non-enriched library (input) as a control. Comparison of input and enriched libraries showed that 5-hmC in exons is twice as frequent as in introns. Functional analysis indicated that 5-hmC abundance is associated with genes that are involved in the adenylate cyclase-activating G-protein-coupled receptor signaling pathway, molting cycles, morphogenesis and cell fate determination. Genes that lack 5-hmC tend to be involved in the regulation of the transforming growth factor beta receptor signaling pathway and in many mRNA-related processes. Our results suggest that epigenetic modifications are present in the genome of D. pulex and most likely are involved in the regulation of gene expression of this crustacean.

Deciphering the Biosynthetic Origin of L-allo-Isoleucine.

The nonproteinogenic amino acid L-allo-isoleucine (L-allo-Ile) is featured in an assortment of life forms comprised of, but not limited to, bacteria, fungi, plants and mammalian systems including Homo sapiens. Despite its ubiquity and functional importance, the specific origins of this unique amino acid have eluded characterization. In this study, we describe the discovery and characterization of two enzyme pairs consisting of a pyridoxal 5'-phosphate (PLP)-linked aminotransferase and an unprecedented isomerase synergistically responsible for the biosynthesis of L-allo-Ile from L-isoleucine (L-Ile) in natural products. DsaD/DsaE from the desotamide biosynthetic pathway in Streptomyces scopuliridis SCSIO ZJ46, and MfnO/MfnH from the marformycin biosynthetic pathway in Streptomyces drozdowiczii SCSIO 10141 drive L-allo-Ile generation in each respective system. In vivo gene inactivations validated the importance of the DsaD/DsaE pair and MfnO/MfnH pair in L-allo-Ile unit biosynthesis. Inactivation of PLP-linked aminotransferases DsaD and MfnO led to significantly diminished desotamide and marformycin titers, respectively. Additionally, inactivation of the isomerase genes dsaE and mfnH completely abolished production of all L-allo-Ile-containing metabolites in both biosynthetic pathways. Notably, in vitro biochemical assays revealed that DsaD/DsaE and MfnO/MfnH each catalyze a bidirectional reaction between L-allo-Ile and L-Ile. Site-directed mutagenesis experiments revealed that the enzymatic reaction involves a PLP-linked ketimine intermediate and uses an arginine residue from the C-terminus of each isomerase to epimerize the amino acid β-position. Consequently, these data provide important new insight into the origins of L-allo-Ile in natural products with medicinal potential and illuminate new possibilities for biotool development.

Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles.

Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

Enhanced Diffusion of Glucose-Fueled Janus Particles

The search for biocompatible fuels to induce autonomous motion in particles is a long-standing challenge in the field of nanorobotics. Hydrogen peroxide (H2O2) is the most utilized fuel for micro/nanomotors, although its cytotoxicity impedes its application in a biomedical context. Biocompatibility not only involves the adjustment of the motor, which should convert the energy from a stable compound into locomotion, but also requires new fabrication methods and the generation of nontoxic products resulting from fuel consumption. To address this challenge, we present the assembly and enhanced diffusion of sub-micron-sized Janus particles that feature one hemisphere decorated with the enzyme pair glucose oxidase and catalase and the use of glucose as fuel. It is found that the colloids exhibit glucose-concentration-dependent enhanced diffusion behavior, thus bringing the concept of nanomachines closer to use in biomedical applications.

Identification of tissue-restricted bioreaction suitable for in vivo targeting by fluorescent substrate library-based enzyme discovery.

Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positive candidate proteins were identified by diced electrophoresis gel assay followed by peptide mass fingerprinting. We discovered that pyruvyl anilide is specifically hydrolyzed by carboxylesterase 2 (CES2), which is predominantly localized in the liver and kidney. We show that the pyruvyl targeting group/CES2 enzyme pair can be used to deliver the 7-amino-4-methylcoumarin fluorophore specifically to the liver and kidney in vivo. Our screening approach should be useful to find other masking group/enzyme pairs suitable for development of fluorescent substrates and prodrugs.

Insight into the genomic diversity and relationship of Astragalus glycyphyllos symbionts by RAPD, ERIC-PCR, and AFLP fingerprinting.

We assessed the genomic diversity and genomic relationship of 28 Astragalus glycyphyllos symbionts by three methodologies based on PCR reaction, i.e., RAPD, ERIC-PCR, and AFLP. The AFLP method with one PstI restriction enzyme and selective PstI-GC primer pair had a comparable discriminatory power as ERIC-PCR oneand these fingerprinting techniques distinguished among the studied 28 A. glycyphyllos symbionts 18 and 17 genomotypes, respectively. RAPD method was less discriminatory in the genomotyping of rhizobia analyzed and it efficiently resolved nine genomotypes. The cluster analysis of RAPD, ERIC-PCR, and AFLP profiles resulted in a generally similar grouping of the test strains on generated dendrograms supporting a great potential of these DNA fingerprinting techniques for study of genomic polymorphism and evolutionary relationship of A. glycyphyllos nodulators. The RAPD, ERIC-PCR, and AFLP pattern similarity coefficients between A. glycyphyllos symbionts studied was in the ranges 8–100, 18–100, and 23-100 %, respectively.

SIRT3-dependent GOT2 acetylation status affects the malate-aspartate NADH shuttle activity and pancreatic tumor growth.

The malate-aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate-aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD(+) redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth invivo. Finally, we show that GOT2K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate-aspartate NADH shuttle activity and oxidative protection.