L-lactate Dehydrogenase Research Articles

Identification and characterization of cadmium stress-related LncRNAs from Betula platyphylla

Cadmium (Cd) is one of the most serious global environmental pollutants, which inhibits plant growth and interferes with their physiological processes. However, there have been few studies on the involvement of long noncoding RNAs (lncRNAs) in Cd tolerance. In the present study, we identified the lncRNAs from Betula platyphylla (birch) that respond to Cd stress. Thirty lncRNAs that were differentially expressed under Cd treatment were identified, including 16 upregulated and 14 downregulated lncRNAs. Nine differentially regulated lncRNAs were selected for further characterization. These lncRNAs were transiently overexpressed in birch plants to determine their roles in Cd tolerance. Among them, two lncRNAs conferred Cd tolerance and two induced sensitivity to Cd stress. We further determined the Cd tolerance of four target genes of the lncRNAs involved in Cd tolerance, including l-lactate dehydrogenase A (LDHA),heat shock protein (HSP18.1), yellow stripe-like protein (YSL9), and H/ACA ribonucleoprotein complex subunit 2-like protein (HRCS2L). Among them, HSP18.1 and LDHA showed improved tolerance to Cd stress, whereas the other two genes did not appear to be involved in Cd tolerance. These results suggested that lncRNAs can up- or downregulate their target genes to improve Cd tolerance. These results increase our understanding of lncRNA-mediated Cd tolerance.

Major role of lactate dehydrogenase D-LDH1 for the synthesis of lactic acid in Fructobacillus tropaeoli CRL 2034.

The enzymes D- and L-lactate dehydrogenase are involved in the reduction of pyruvate to D(+)- and L(-)-lactate, respectively. The fig-origin strain Fructobacillus tropaeoli CRL 2034 produces D- and L-lactic acids in a 9:1 ratio. In this work, two D-ldh (ldh1 and ldh2) and one L-ldh (ldh3) genes were found in the CRL 2034 genome. ldh1 and ldh2 are homologous (79% identity) and organized as contiguous operons, each gene containing 996 base pair (bp) and encoding for a 331-amino acid (aa) protein (74% identity). In contrast, ldh3 is a 927-bp gene coding for a 308-aa protein. The identity between ldh1/ldh2 and ldh3 was lower than 48%. To elucidate the role of these genes in the synthesis of lactic acid by the Fructobacillus strain, plasmid insertion mutants in each gene were generated and characterized. The growth kinetic parameters were affected only in CRL2034 ldh1::pRV300 cells, this mutant showing the lowest total lactic acid production (4.50 ± 0.15 versus 6.36 ± 0.67 g/L of wild-type strain), with a D/L ratio of 7.1:2.9. These results showed that the ldh1 gene is primarily responsible for lactic acid production by the studied strain. A comparative analysis among strains of the five Fructobacillus species revealed that the identity of D-LDH proteins was higher than 70%, while the identity of L-LDH was over 60%. Finally, phylogenetic analysis of D- and L-LDHs revealed that only D-LDH phylogeny was consistent to the phylogenetic evolution among Fructobacillus and evolutionarily related genera. Key Points •F. tropaeoli CRL 2034 harbors three ldh genes in its genome. •ldh1 and ldh2 encode D-lactate dehydrogenase; ldh3 encodes L-lactate dehydrogenase. •Gene ldh1 plays the major role in lactic acid production by strain CRL 2034. •Fructobacillus D-LDH phylogeny was consistent to phylogenetic evolution.

Metabolically engineered Lactobacillus gasseri JCM 1131 as a novel producer of optically pure L- and D-lactate.

High-quality environmentally-friendly bioplastics can be produced by mixing poly-L-lactate with poly-D-lactate. On an industrial scale, this process simultaneously consumes large amounts of both optically pure lactate stereoisomers. However, because optimal growth conditions of L-lactate producers often differ from those of D-lactate producers, each stereoisomer is produced in a specialised facility, which raises cost and lowers sustainability. To address this challenge, we metabolically engineered Lactobacillus gasseri JCM 1131T, a bioprocess-friendly and genetically malleable strain of homofermentative lactic acid bacterium, to efficiently produce either pure L- or pure D-lactate under the same bioprocess conditions. Transformation of L. gasseri with plasmids carrying additional genes for L- or D-lactate dehydrogenases failed to affect the ratio of produced stereoisomers, but inactivation of the endogenous genes created strains which yielded 0.96g of either L- or D-lactate per gram of glucose. In this study, the plasmid pHBintE, routinely used for gene disruption in Bacillus megaterium, was used for the first time to inactivate genes in lactobacilli. Strains with inactivated genes for endogenous lactate dehydrogenases efficiently fermented sugars released by enzymatic hydrolysis of alkali pre-treated wheat straw, an abundant lignocellulose-containing raw material, producing 0.37-0.42g of lactate per gram of solid part of alkali-treated wheat straw. Thus, the constructed strains are primed to serve as producers of both optically pure L-lactate and D-lactate in the next-generation biorefineries.

Participation of two general stress response proteins from Xanthomonas citri subsp. citri in environmental stress adaptation and virulence.

Xanthomonas citri subsp. citri (Xcc) is the bacteria responsible for citrus canker. During its life cycle Xcc is found on leaves as epiphyte, where desiccation conditions may occur. In this work, two Xcc genes, XAC0100 and XAC4007, predicted in silico to be involved in general stress response, were studied under salt, osmotic, desiccation, oxidative and freezing stress, and during plant-pathogen interaction. Expression of XAC0100 and XAC4007 genes was induced under these stress conditions. Disruption of both genes in Xcc caused decreased bacterial culturability under desiccation, freezing, osmotic and oxidative stress. Importantly, the lack of these genes impaired Xcc epiphytic fitness. Both Xac0100 and Xac4007 recombinant proteins showed protective effects on Xanthomonas cells subjected to drought stress. Also, Escherichia coli overexpressing Xac4007 showed a better performance under standard culture, saline and osmotic stress and were more tolerant to freezing and oxidative stress than wild type E. coli. Moreover, both Xac0100 and Xac4007 recombinant proteins were able to prevent the freeze-thaw-induced inactivation of L-Lactate dehydrogenase. In conclusion, Xac0100 and Xac4007 have a relevant role as bacteria and protein protectors; and these proteins are crucial to bacterial pathogens that must face environmental stressful conditions that compromise the accomplishment of the complete virulence process.

Abstract A08: Cadmium elicits a differential cytotoxic response in triple-negative breast cancer cells

Abstract Understanding the relationship between heavy metal exposure and triple-negative breast cancer cells may help to elucidate the high breast cancer mortality rate and health disparity in African-American women, as they may be exposed to these heavy metals at higher rates when compared to their Caucasian counterparts. Triple-negative breast vancer (TNBC) accounts for about 15% of all breast cancers and is characterized by cancer cells that lack the expression of estrogen, progesterone, and HER2/Neu receptors. For this reason, TNBC cells do not respond to hormone therapies or HER2/Neu-targeted treatments. The human body utilizes some metals in small amounts as micronutrients; however, metals such as cadmium-2 that are introduced to the body are not needed physiologically. Human exposure to trace metals in higher-than-needed amounts or exposure to nonessential metals can cause damage to the body as they can be toxic or even carcinogenic. Here we expose the TNBC cell lines HCC 1806 and HCC 70 to cadmium-2. While less is known about the long-term carcinogenic potential of cadmium exposure in the broader population and for other organs, evidence suggests its potential association to lung, breast, and endometrial cancer and mortality. Previous lab data revealed that exposure to high concentrations of cadimum-2 ions causes a differential cytotoxic cell death response in TNBC cells and demonstrates different genotoxic targets and implications in their mutagenic potential when HCC 1806 and HCC 70 cell lines were exposed to this ion. Here, the cell lines have been treated with 5 ug/ml of cadmium-2 ion, revealing increased toxicity using the lactate dehydrogenases assay (LDH), decreased viability (MTS assay), and flow cytometry reveals an abrogation of the cell cycle. The LDH assay revealed that the cells exposed to cadmium resulted in a P-value less than 0.0001 and assay data present percent cytotoxicity of 14.13 and 12.58, respectively, for control and treated cells. A similar trend was exhibited in HCC 70, where the LDH assay resulted in a p-value less than 0.0001 and assay data demonstrated a percent cytotoxicity of 11.57 and 6.92, respectively, for control and treated cells. The MTS assay alongside trypan blue exclusion assay demonstrated a trend of decreasing viability among the treated cells. The flow cytometric analysis revealed an increase in the S-phase in the cadmium-treated cells compared to the controls. These results are potentially early indicators of a molecular effect of Cd ions on breast cancer cells that could elucidate the role that cadmium may play in promoting the aggressive behavior of TNBC or could potentially lead to future treatment regimens. Citation Format: Sherette Godfrey, Checo Rorie. Cadmium elicits a differential cytotoxic response in triple-negative breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A08.

Substrate Channeling via a Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase

Substrate channeling studies have frequently failed to provide conclusive results due to poor understanding of this subtle phenomenon. We analyzed the mechanism of NADH-channeling from D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to L-lactate Dehydrogenase (LDH) using enzymes from different cells. Enzyme kinetics studies showed that LDH activity with free NADH and GAPDH-NADH complex always take place in parallel. The channeling is observed only in assays that mimic cytosolic conditions where free NADH concentration is negligible and the GAPDH-NADH complex is dominant. Molecular dynamics and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at the limiting NADH concentrations. Surface calculations showed that positive electric field between the NAD(H) binding sites on LDH and GAPDH tetramers can merge in the LDH-GAPDH complex. NAD(H)-channeling within the LDH-GAPDH complex can be an extension of NAD(H)-channeling within each tetramer. In the case of a transient LDH-(GAPDH-NADH) complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between the off-rates for the LDH-(GAPDH-NADH) complex and the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths.

A computational approach to validate novel drug targets of gentianine from Swertiya chirayita in Plasmodium falciparum

Gentianine is one of the compounds found in the plant Swertiya chirayita that is known for its antimalarial activity. However, its exact molecular mechanism of action is yet to be understood. In our present study, we applied several computational approaches to filter out and determine possible targets of gentianine in Plasmodium falciparum 3D7. Protein-protein networks formed the basis of one of our strategies along with orthologous protein analysis to establish essentiality. Out of 6 essential proteins from unique pathways, haloacid dehalogenase like-hydrolase (PfHAD1), phosphoenolpyruvate carboxykinase (PfPEPCK) and fumarate hydratase (PfFH) were screened as drug targets through this approach. Through our other strategy we established the predicted IC50 (PIC50) value of gentianine with a set of molecular descriptors from 123 Pathogen Box anti-malarial compounds. Afterwards through 2D structural similarity, L-lactate dehydrogenase (PfLDH) was established as another possible target. In our work, we performed in silico docking and analysed the binding of gentianine to the proteins. All of the proteins were reported with favourable binding results and were considered for complex molecular dynamics simulation approach. Our research clears up the molecular mechanism of antimalarial activity of gentianine to some extent paving way for experimental validation of the same in future.

Physiological response of mammary glands to Escherichia coli infection: A conflict between glucose need for milk production and immune response

The mammary immune and physiological responses to distinct mammary-pathogenic E. coli (MPEC) strains were studied. One gland in each of ten cows were challenged intra-mammary and milk composition (lactose, fat, total protein, casein), biochemical (glucose, glucose-6-phosphate (Glu6P), oxalate, malate, lactate, pyruvate and citrate, malate and lactate dehydrogenases, lactate dehydrogenase (LDH), nitrite, lactic peroxidase, catalase, albumin, lactoferrin, immunoglobulin) and clotting parameters were followed for 35 days post-challenge. Challenge lead to clinical acute mastitis, with peak bacterial counts in milk at 16–24 h post-challenge. Biochemical and clotting parameters in milk reported were partially in accord with lipopolysaccharide-induced mastitis, but increased Glu6P and LDH activity and prolonged lactate dehydrogenase and Glu6P/Glu alterations were found. Some alterations measured in milk resolved within days after challenge, while others endured for above one month, regardless of bacterial clearance, and some reflected physiological responses to mastitis such as the balance between aerobic and anaerobic metabolism (citrate to lactate ratios). The results suggest that E. coli mastitis can be divided into two stages: an acute, clinical phase, as an immediate response to bacterial infection in the mammary gland, and a chronic phase, independent of bacteria clearance, in response to tissue damage caused during the acute phase.

Clinical and Diagnostic Significance of Lactate Dehydrogenase and Its Isoenzymes in Animals.

Lactate dehydrogenase (LDH) is widely distributed enzyme in cells of various living systems where it is involved in carbohydrate metabolism catalyzing interconversion of lactate and pyruvate with NAD+/NADH coenzyme system. Cells of tissues are direct source of lactate dehydrogenase isoenzymes that are naturally distributed in blood plasma/serum of animals and humans producing characteristic profile. This profile depends on intracellular isoenzyme concentration in all tissues that contribute to the common pool of lactate dehydrogenases in plasma/serum as a consequence of natural cell degradation. LDH is widely distributed in the body, high activities are found in the heart, liver, skeletal muscle, kidney, and erytrocytes, whereas lesser amounts are found in the lung, smooth muscle, and brain. Because of its widespread activities in numerous body tissues, LDH is elevated in a variety of disorders. There are many conditions that contribute to increased activity of LDH. An elevated total LDH value is a rather nonspecific finding. Therefore, LDH assays assume a more clinical significance when separated into isoenzyme fractions. The activity of LDH and its serum and tissue patterns and composition show great variations between the species. These differences do not allow using catalytic activities of LDH isoenzymes from one species to another. Instead, the pattern of serum LDH isoenzymes should be interpreted in respect to its species origin that is important in particular in veterinary medicine. Determination of total LDH activity and its isoenzyme pattern in serum of mammals had become one of the biochemical indicators in the assessment of organ disorders. When the content of cells is released from tissue to plasma, as on cell injury, the LDH isoenzyme pattern of the serum changes in favour of the profile of the affected organ (tissue) that can be used in the diagnostic practice.

Deciphering the d-/l-lactate-producing microbiota and manipulating their accumulation during solid-state fermentation of cereal vinegar

Symphony orchestra of multi-microorganisms characterizes the solid-state acetic acid fermentation process of Chinese cereal vinegars. Lactate is the predominant non-volatile acid and plays indispensable roles in flavor formation. This study investigated the microbial consortia driving the metabolism of D-/l-lactate during fermentation. Sequencing analysis based on D-/l-lactate dehydrogenase genes demonstrated that Lactobacillus (relative abundance: > 95%) dominated the production of both d-lactate and l-lactate, showing species-specific features between the two types. Lactobacillus helveticus (>65%) and L. reuteri (~80%) respectively dominated l- and d-lactate-producing communities. D-/l-lactate production and utilization capabilities of eight predominant Lactobacillus strains were determined by culture-dependent approach. Subsequently, D-/l-lactate producer L. plantarum M10-1 (d:l ≈ 1:1), l-lactate producer L. casei 21M3-1 (D:L ≈ 0.2:9.8) and D-/l-lactate utilizer Acetobacter pasteurianus G3-2 were selected to modulate the metabolic flux of D-/l-lactate of microbial consortia. The production ratio of D-/l-lactate was correspondingly shifted coupling with microbial consortia changes. Bioaugmentation with L.casei 21M3-1 merely enhanced l-lactate production, displaying ~4-fold elevation at the end of fermentation. Addition of L.plantarum M10-1 twice increased both D- and l-lactate production, while A. pasteurianus G3-2 decreased the content of D-/l-isomer. Our results provided an alternative strategy to specifically manipulate the metabolic flux within microbial consortia of certain ecological niches.

Proteomic identification of aerobic glycolysis as a potential metabolic target for methylglyoxal in adipocytes

Obesity is often accompanied by metabolic changes in adipocytes that are closely associated with metabolic disease. Although high sugar consumption contributes to obesity, it may also directly affect adipocytes by increasing the rate of glycolysis and formation of the glycolytic by-product methylglyoxal (MG). MG is a reactive dicarbonyl that irreversibly damages proteins and other cellular components. Although the accumulation of MG is clinically associated with hyperglycemia and diabetic complications, a better understanding of how proteins are regulated by MG is needed to evaluate its role in the pathogenesis of metabolic disease. Because adipocytes rely heavily on glycolysis for glucose disposal, we hypothesized that prolonged MG treatment at nontoxic concentrations would impact the landscape of proteins involved in glucose metabolism. To test this hypothesis, we treated 3T3-L1 adipocytes with MG (100 μmol/L) and used comparative proteomics to assess the effects. We identified 25 differentially expressed proteins in adipocytes treated with MG compared to the control. Our results suggested that MG induced metabolic changes typically associated with aerobic glycolysis, including a lowered expression of proteins involved in oxidative metabolism and increased expression of the glycolytic enzymes L-lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The detection of increased lactate secreted into the culture media of adipocytes treated with MG further supported these findings, as did gene expression analysis. In summary, these results indicate MG as a metabolic contributor to aerobic glycolysis in adipocytes, a potential adaptive response to increased glucose flux which over time could lead to permanent metabolic changes.

Buddleja asiatica Lour. Derived Phytochemicals against Staphylococcus aureus Causing Skin Diseases

Phytochemicals from Buddleja asiatica plant extract are traditionally used to cure skin diseases. It is caused by Staphylococcus aureus. Molecular docking method applied using “Biovia Discovery Studio”. “High positive values of -CDOCKER energy and -CDOCKER interaction energy” suggested that lignoceric acid can effectively deactivate the L-lactate dehydrogenase enzyme thereby interrupting the life cycle of the organism.

Abstract B072: Cadmium elicits a differential cytotoxic response in triple-negative breast cancer cells

Abstract Understanding the relationship between heavy metal exposure and triple-negative breast cancer cells may help to elucidate the high breast cancer mortality rate and health disparity in African-American women, as they may be exposed to these heavy metals at higher rates and/or their cells may react more negatively to the heavy metals when compared to their Caucasian counterparts. Triple-negative breast cancer (TNBC) accounts for about 15% of all breast cancers and is characterized by cancer cells that lack the expression of estrogen, progesterone, and HER2/Neu receptors. For this reason, TNBC cells do not respond to hormone therapies or HER2/Neu targeted treatments. The human body utilizes some metals in small amounts as micronutrients; however, many metals such as cadmium-2, chromium-6, arsenic, lead, and mercury that are introduced to the body are not needed physiologically. Human exposure to trace metals in higher-than-needed amounts or exposure to nonessential metals can cause damage to the body as they can be toxic or even carcinogenic. Here we expose the TNBC cell line HCC 1806 to Cadmium-2. While less is known about the long-term carcinogenic potential of cadmium exposure in the broader population and for other organs, evidence suggests its potential association to lung, breast, and endometrial cancer and mortality. Previous lab data revealed that exposure to high concentrations of Cadimum-2 ions causes a differential cytotoxic cell death response in TNBC cells and demonstrates different genotoxic targets and implications in their mutagenic potential when HCC 1806 cell lines were exposed to this ion. Here, the cell line HCC 1806 has been treated with 5 ug/ml of Cadmium-2 ion, revealing increased toxicity using the lactate dehydrogenases assay (LDH), decreased viability (MTS assay), and flow cytometry reveals an abrogation of the cell cycle. The LDH assay revealed that the cells exposed to cadmium resulted in a P-value less than 0.0001 and assay data present percent cytotoxicity of 5.1 and 10.7, respectively, for control and treated cells. The MTS assay alongside trypan blue exclusion assay demonstrated a trend of decreasing viability among the treated cells. The flow cytometric analysis revealed an increase in the S-Phase in the cadmium treated cells compared to the controls. These results are potentially early indicators of a molecular effect of Cd ions on breast cancer cells that could elucidate the role that cadmium may play in promoting the aggressive behavior of TNBC or could potentially lead to future treatment regiments. Citation Format: Sherette Godfrey, Checo Rorie. Cadmium elicits a differential cytotoxic response in triple-negative breast cancer cells [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B072.

Expression of a Lactobacillus casei L-lactate dehydrogenase mutant in Pichia pastoris for asymmetric reduction of phenylpyruvate

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.

Evaluation of Product Distribution in Chemostat and Batch Fermentation in Lactic Acid-Producing Komagataella phaffii Strains Utilizing Glycerol as Substrate.

Lactic acid is the monomeric unit of polylactide (PLA), a bioplastic widely used in the packaging, automotive, food, and pharmaceutical industries. Previously, the yeast Komagataella phaffii was genetically modified for the production of lactate from glycerol. For this, the bovine L-lactate dehydrogenase- (LDH)-encoding gene was inserted and the gene encoding the pyruvate decarboxylase (PDC) was disrupted, resulting in the GLp strain. This showed a yield of 67% L-lactic acid and 20% arabitol as a by-product in batches with oxygen limitation. Following up on these results, the present work endeavored to perform a detailed study of the metabolism of this yeast, as well as perturbing arabitol synthesis in an attempt to increase lactic acid titers. The GLp strain was cultivated in a glycerol-limited chemostat at different dilution rates, confirming that the production of both lactic acid and arabitol is dependent on the specific growth rate (and consequently on the concentration of the limiting carbon source) as well as on the oxygen level. Moreover, disruption of the gene encoding arabitol dehydrogenase (ArDH) was carried out, resulting in an increase of 20% in lactic acid and a 50% reduction in arabitol. This study clarifies the underlying metabolic reasons for arabitol formation in K. phaffii and points to ways for improving production of lactic acid using K. phaffii as a biocatalyst.

Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions

The oxygen-limiting condition promotes the accumulation of ployhydroxybutyrate (PHB) in C. necator H16, while the growth of which is restricted. Under autotrophic culture using carbon dioxide, hydrogen, and oxygen as substrates, the oxygen concentration below 6.9% (v/v) in the mixture is considered as a safe condition. It also expected to achieve cell rapid growth and large accumulation of PHB simultaneously under the oxygen-limiting condition in C. necator H16. In this study, a metabolically engineered strain capable of both rapid growth and large accumulation of PHB under oxygen-limiting conditions was constructed based on the transcriptomic analysis. In the comparative transcriptomic analysis, the genes related to energy-generating of C. necator H16 at autotrophic culture were downregulated under oxygen-limiting conditions (3%, v/v). Besides, the genes related to the key intermediates (pyruvate and acetyl-CoA) metabolism in PHB biosynthetic pathway were analyzed. Most of which were downregulated, except the genes ldh, iclA, and ackA2 respectively encoding L-lactate dehydrogenase, isocitrate lyase, and acetate kinase were upregulated under oxygen-limiting conditions (3%, v/v). The Vitreoscilla hemoglobin (VHb) has the ability to promote aerobic metabolism and energy generation. To promote the bacterium growth and improve the energy generation in C. necator H16 under oxygen-limiting conditions, the VHb gene was introduced into C. necator H16 with the optimized promoter PphaC1-j5. Moreover, VHb was localized to the periplasmic space of the bacterium by the traction of membrane-bound hydrogenase (MBH) signal peptide. By optimizing the knockout of different genes, it was found that knockout of ldh can improve PHB production and reduce the by-products. Finally, a recombinant strain Reh01 (p2M-pj-v) was constructed by heterologous expression of vgb and ldh knockout in C. necator H16. Compared with the control (Reh (p2)) under oxygen-limiting conditions (3%, v/v), the dry cell weight (DCW), PHB content, and PHB production of Reh01 (p2M-pj-v) increased by 31.0%, 30.9%, and 71.5%, respectively. From the perspectives of transcriptome and metabolic engineering, the work provides new ideas to achieve rapid cell growth and large PHB accumulation in C. necator under oxygen-limiting and autotrophic conditions.

Pressure tolerance of deep-sea enzymes can be evolved through increasing volume changes in protein transitions: a study with lactate dehydrogenases from abyssal and hadal fishes.

We explore the principles of pressure tolerance in enzymes of deep‐sea fishes using lactate dehydrogenases (LDH) as a case study. We compared the effects of pressure on the activities of LDH from hadal snailfishes Notoliparis kermadecensis and Pseudoliparis swirei with those from a shallow‐adapted Liparis florae and an abyssal grenadier Coryphaenoides armatus. We then quantified the LDH content in muscle homogenates using mass‐spectrometric determination of the LDH‐specific conserved peptide LNLVQR. Existing theory suggests that adaptation to high pressure requires a decrease in volume changes in enzymatic catalysis. Accordingly, evolved pressure tolerance must be accompanied with an important reduction in the volume change associated with pressure‐promoted alteration of enzymatic activity ( ΔVPP∘ ). Our results suggest an important revision to this paradigm. Here, we describe an opposite effect of pressure adaptation—a substantial increase in the absolute value of ΔVPP∘ in deep‐living species compared to shallow‐water counterparts. With this change, the enzyme activities in abyssal and hadal species do not substantially decrease their activity with pressure increasing up to 1–2 kbar, well beyond full‐ocean depth pressures. In contrast, the activity of the enzyme from the tidepool snailfish, L. florae, decreases nearly linearly from 1 to 2500 bar. The increased tolerance of LDH activity to pressure comes at the expense of decreased catalytic efficiency, which is compensated with increased enzyme contents in high‐pressure‐adapted species. The newly discovered strategy is presumably used when the enzyme mechanism involves the formation of potentially unstable excited transient states associated with substantial changes in enzyme–solvent interactions.

Interrogating the Lactate Dehydrogenase Tetramerization Site Using (Stapled) Peptides.

Lactate dehydrogenases (LDHs) are tetrameric enzymes of major significance in cancer metabolism as well as promising targets for cancer therapy. However, their wide and polar catalytic sites make them a challenging target for orthosteric inhibition. In this work, we conceived to target LDH tetramerization sites with the ambition of disrupting their oligomeric state. To do so, we designed a protein model of a dimeric LDH-H. We exploited this model through WaterLOGSY nuclear magnetic resonance and microscale thermophoresis for the identification and characterization of a set of α-helical peptides and stapled derivatives that specifically targeted the LDH tetramerization sites. This strategy resulted in the design of a macrocyclic peptide that competes with the LDH tetramerization domain, thus disrupting and destabilizing LDH tetramers. These peptides and macrocycles, along with the dimeric model of LDH-H, constitute promising pharmacological tools for the de novo design and identification of LDH tetramerization disruptors. Overall, our study demonstrates that disrupting LDH oligomerization state by targeting their tetramerization sites is achievable and paves the way toward LDH inhibition through this novel molecular mechanism.

Discovery of Potential Plasma Biomarkers for Tuberculosis in HIV-Infected Patients by Data-Independent Acquisition-Based Quantitative Proteomics.

PurposeTuberculosis (TB) is the leading cause of mortality in individuals infected with human immunodeficiency virus (HIV), yet the methods for detecting Mycobacterium tuberculosis at an early stage remain insensitive or ineffective. This study aimed to discover plasma biomarkers for distinguishing HIV-TB coinfected individuals from HIV individuals without TB (HIV-nonTB).Patients and MethodsA total of 200 Chinese HIV-positive patients were recruited, 100 each for HIV-nonTB group and HIV-TB group. Plasma proteomic profiles were analyzed for 50 patients each in both groups, using data-independent acquisition (DIA)-mass spectrometry-based proteomics. Differently expressed proteins were revealed with ridge regression analysis. Enzyme-linked immunosorbent assay (ELISA) analyses were performed for further validation in other 100 patients.ResultsDIA-mass spectrometry revealed 13 upregulated and 33 downregulated proteins in the HIV-TB group. AMACR (α-methylacyl-CoA racemase), LDHB (L-lactate dehydrogenase B chain), and RAP1B (Ras-related protein Rap-1b) were selected for building a diagnostic model, for which the receiver operation characteristic curve had under areas of 0.99 and 0.89 testing with proteomics data (sensitivity = 92%, specificity = 100%) and ELISA data (sensitivity = 76%, specificity = 92%), respectively.ConclusionThe combination of AMACR, LDHB, and RAP1B proteins may serve as a potential marker of TB in HIV-infected patients.

Lactate and Acidity in the Cancer Microenvironment

Fermentative glycolysis, an ancient evolved metabolic pathway, is exploited by rapidly growing tissues and tumors but also occurs in response to the nutritional and energetic demands of differentiated tissues. The lactic acid it produces is transported across cell membranes through reversible H+/lactate−symporters (MCT1 and MCT4) and is recycled in organs as a major metabolic precursor of gluconeogenesis and an energy source. Concentrations of lactate in the tumor environment, investigated utilizing an induced metabolic bioluminescence imaging (imBI) technique, appear to be dominant biomarkers of tumor response to irradiation and resistance to treatment. Suppression of lactic acid formation by genetic disruption of lactate dehydrogenases A and B in aggressive tumors reactivated OXPHOS (oxidative phosphorylation) to maintain xenograft tumor growth at a halved rate. In contrast, disruption of the lactic acid transporters MCT1/4 suppressed glycolysis, mTORC1, and tumor growth as a result of intracellular acidosis. Furthermore, the global reduction of tumor acidity contributes to activation of the antitumor immune responses, offering hope for future clinical applications.