Design and evolution of an enzyme with a non-canonical organocatalytic mechanism.

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

Acute effects of oral mesna administration on the full amino acid profile and 3-methylhistidine: secondary results from the CYLOB dose-finding study.

Plasma total cysteine (tCys) is strongly associated with fat mass in humans. Mesna lowers plasma tCys in a dose-dependent manner, but it is not known whether it interferes with metabolism of other amino acids or protein. In this Phase-1 study, we show that a single dose of mesna administered at 400, 800, 1200 or 1600 mg to 6-7 individuals per dose only slightly affects amino acid profiles, with increases in plasma valine across dose levels. There were no effects of mesna on 3-methylhistidine, a marker of protein breakdown.

Variation in protein metabolism biomarkers during the transition period and associations with health, colostrum quality, reproduction, and milk production traits in Holstein cows.

The aims of this study were to assess (1) the variation of protein metabolism biomarkers and factors affecting them during the transition period, (2) the association of each biomarker with skeletal muscle reserves and their changes, and (3) the association of these biomarkers with postpartum health, colostrum quality, reproduction, and milk production. For this purpose, 238 multiparous Holstein cows from 6 herds were used in a prospective cohort study. Plasma concentrations of 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH) and serum concentrations of total protein (TP), albumin (ALB), urea nitrogen (BUN), and creatinine (SCR) were determined for each cow at -21, -7, 7, 21, and 28 d relative to calving. Clinical diseases were recorded during the first 28 d postcalving, and presence of subclinical ketosis (scKET) was investigated at 7 and 21 d. Colostrum quality was estimated by Brix refractometry. Reproduction data by 150 d in milk (DIM) and milk production records were also available. Linear mixed models including the fixed effects of time point, herd, parity, body condition score (-21 d), duration of dry period and postparturient diseases were fitted to assess the variation in each biomarker's concentration. The association between the biomarkers' concentration during the prepartum period with the odds for each postparturient disease and for a combined trait (CD_1-28), defined as the presence of at least one clinical condition during the first 28 d after calving, were assessed with separate binary logistic models for time points -21 d and -7 d. The relationship of each biomarker's concentration with longissimus dorsi thickness (LDT) and the changes in LDT (ΔLDT) was assessed with pairwise correlations. Separate general linear models were used to assess the association of each biomarker with colostrum Brix values and milk production traits. Finally, the associated hazard for first artificial insemination (AI) and for pregnancy by 150 DIM (PREG_150DIM) was assessed with Cox proportional hazard models, whereas odds for pregnancy to the first AI (PREG_1stAI) were assessed with binary logistic models. The level of 3-MH was affected mainly by herd, time points, and their interaction. Higher 3-MH was associated with increased odds for metritis and CD_1-28, increased hazard for PREG_150 DIM and with increased milk production. 1-Methylhistidine was affected mainly by herd, scKET and occurrence of displaced abomasum. Higher 1-MH was associated with better colostrum quality, increased odds for scKET, increased hazard for first AI by 150 DIM and with decreased milk production. Both 3-MH and 1-MH were weakly to moderately negatively correlated with LDT and moderately to strongly negatively correlated with ΔLDT at the corresponding time periods. Additionally, higher TP was associated with increased odds for metritis and CD_1-28 and increased milk production, while higher ALB was associated with increased odds for scKET and increased milk production. Moreover, higher BUN was associated with decreased odds for scKET, increased odds for PREG_1stAI and increased milk production. Higher SCR was associated with decreased odds for retained fetal membranes, metritis, and CD_1-28. Periparturient protein metabolism is significantly associated with postpartum health, colostrum quality, reproduction, and milk production; mechanisms involved require further investigation.

Greater plasma essential amino acids and lower 3-methylhistidine with higher protein intake during endurance training: a randomised control trial.

Endurance exercise alters amino acid (AA) metabolism that necessitates greater AA intake in the post exercise recovery period to support recovery. Thus, daily AA ingestion during a period of endurance training may affect the metabolically active plasma free AA pool, which is otherwise maintained during periods of inadequate protein intake by the breakdown of skeletal muscle proteins. Nine endurance-trained males completed a 4-day running protocol (20 km, 5 km, 10 km and 20 km on days 1-4, respectively) on three occasions with a controlled diet providing different protein intakes [0.94(LOW), 1.20(MOD) or 1.83gprotein kgbody mass-1 day-1 (HIGH)]. Urine collected over 24 h on day-4 and plasma collected after an overnight fast on day-5 were analyzed for free AA (plasma) and 3-methylhistidine (3MH; plasma and urine), a marker of myofibrillar protein breakdown. There was an effect of protein intake (HIGH > MOD/LOW; P < 0.05) on fasted plasma essential AA, branched chain AA and 3MH but no effect on 24-h urinary 3-MH excretion. Consuming a previously determined optimal daily protein intake of 1.83 g kg-1 day-1 during endurance training maintains fasted plasma free AA and may attenuate myofibrillar protein catabolism, although this latter effect was not detected in 24-h urinary excretion. The maintenance of the metabolically active free plasma AA pool may support greater recovery from exercise and contribute to the previously determined greater whole-body net protein balance in this athletic population. TRN: NCT02801344 (June 15, 2016).

siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9.

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-ß-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC-tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.

PHD3 mediates denervation skeletal muscle atrophy through Nf-κB signal pathway.

Skeletal muscle is the largest organ of the body, the development of skeletal muscle is very important for the health of the animal body. Prolyl hydroxylases (PHDs) are the classical regulator of the hypoxia inducible factor (HIF) signal pathway, many researchers found that PHDs are involved in the muscle fiber type transformation, muscle regeneration, and myocyte differentiation. However, whether PHDs can impact the protein turnover of skeletal muscle is poorly understood. In this study, we constructed denervated muscle atrophy mouse model and found PHD3 was highly expressed in the atrophic muscles and there was a significant correlation between the expression level of PHD3 and skeletal muscle weight which was distinct from PHD1 and PHD2. Then, the similar results were getting from the different weight muscles of normal mice. To further verify the relationship between PHD3 and skeletal muscle protein turnover, we established a PHD3 interference model by injecting PHD3 sgRNA virus into tibialis anterior muscle (TA) muscle of MCK-Cre-cas9 mice and transfecting PHD3 shRNA lentivirus into primary satellite cells. It was found that the Knock-out of PHD3 in vivo led to a significant increase in muscle weight and muscle fiber area (P < .05). Besides, the activity of protein synthesis signal pathway increased significantly, while the protein degradation pathway was inhibited evidently (P < .05). In vitro, the results of 5-ethynyl-2'-deoxyuridine (EdU) and tetramethylrhodamine ethyl ester (TMRE) fluorescence detection showed that PHD3 interference could lead to a decrease in cell proliferation and an increase of cell apoptosis. After the differentiation of satellite cells, the production of puromycin in the interference group was higher than that in the control group, and the content of 3-methylhistidine in the interference group was lower than that in the control group (P < .05) which is consistent with the change of protein turnover signal pathway in the cell. Mechanistically, there is an interaction between PHD3, NF-κB, and IKBα which was detected by immunoprecipitation. With the interfering of PHD3, the expression of the inflammatory signal pathway also significantly decreased (P < .05). These results suggest that PHD3 may affect protein turnover in muscle tissue by mediating inflammatory signal pathway. Finally, we knocked out PHD3 in denervated muscle atrophy mice and LPS-induced myotubes atrophy model. Then, we found that the decrease of PHD3 protein level could alleviate the muscle weight and muscle fiber reduction induced by denervation in mice. Meanwhile, the protein level of the inflammatory signal pathway and the content of 3-methylhistidine in denervated atrophic muscle were also significantly reduced (P < .05). In vitro, PHD3 knock-out could alleviate the decrease of myotube diameter induced by LPS, and the expression of protein synthesis pathway was also significantly increased (P < .05). On the contrary, the expression level of protein degradation and inflammatory signal pathway was significantly decreased (P < .05). Through these series of studies, we found that the increased expression of PHD3 in denervated muscle might be an important regulator in inducing muscle atrophy, and this process is likely to be mediated by the inflammatory NF-κB signal pathway.

3-methylhistidine and clinical outcomes in maintenance haemodialysis patients.

BACKGROUND: Chronic kidney disease is an important contributor to morbidity and mortality. 3-methylhistidine (3-MH) is the by-product of actin and myosin degradation reflecting skeletal muscle turnover. Markedly elevated 3-MH levels have been documented in uraemic patients, but the interpretation of high 3-MH concentration in maintenance haemodialysis (MHD) patients remains unclear. Indeed, it is not known whether elevated serum 3-MH levels are a marker of excessive muscle catabolism or a better lean tissue mass. Here, we evaluated the association between serum 3-MH levels and clinical outcomes in these patients. METHODS: Serum 3-MH concentration was measured by reverse-phase liquid chromatography/tandem mass spectrometry in a cohort of MHD patients. We analysed the relationships between various clinical/laboratory indices, lean tissue mass measured by bioimpedance spectroscopy, mortality and cardiovascular (CV) events. RESULTS: Serum 3-MH concentration was positively correlated with serum albumin, normalized protein catabolic rate (nPCR), simplified creatinine index (SCI) and lean tissue mass. Of 291 MHD patients, during a mean follow-up of 847 days, 91 patients died and 101 patients experienced a CV event. Survival was significantly better in patients with high 3-MH concentrations (P = .002). A higher level of 3-MH was also associated with a lower CV mortality and lower incidence of CV events (P = .015 and P < .001, respectively). Low serum 3-MH levels remained significantly associated with CV events but not with mortality after adjustment for demographic, metabolic and CV risk factors. CONCLUSION: Elevated serum 3-MH concentration appears to be a marker of better lean tissue mass and nutritional status. Low serum 3-MH is a robust and independent predictor of CV events in the MHD population.

A Survey on the Distribution of Ovothiol and ovoA Gene Expression in Different Tissues and Cells: A Comparative Analysis in Sea Urchins and Mussels.

Ovothiols are histidine-derived thiols produced by a variety of marine invertebrates, protists and bacteria. These compounds, which are among the strongest natural antioxidants, are involved in controlling the cellular redox balance due to their redox exchange with glutathione. Although ovothiols were initially reported as protective agents against environmental stressors, new evidence suggests that they can also act as pheromones and participate in fundamental biological processes such as embryogenesis. To get further insight into the biological roles of ovothiols, we compared ovothiol biosynthesis in the sea urchin Paracentrotus lividus and in the mussel Mytilus galloprovincialis, the two species that represent the richest sources of these compounds among marine invertebrates. Ovothiol content was measured in different tissues and in the immune cells from both species and the expression levels of ovoA, the gene responsible for ovothiol biosynthesis, was inferred from publicly available transcriptomes. A comparative analysis of ovothiol biosynthesis in the two species allowed the identification of the tissues and cells synthesizing the metabolite and highlighted analogies and differences between sea urchins and mussels. By improving our knowledge on the biological roles of ovothiols and pointing out the existence of sustainable natural sources for their isolation, this study provides the basis for future biotechnological investigations on these valuable compounds.

Climbing Performance in U23 and Professional Cyclists during a Multi-stage Race.

The aim of this study was to analyze climbing performance across two editions of a professional multistage race, and assess the influence of climb category, prior workload, and intensity measures on climbing performance in U23 and professional cyclists. Nine U23 cyclists (age 20.8±0.9 years) and 8 professional cyclists (28.1±3.2 years) participated in this study. Data were divided into four types: overall race performance, climb category, climbing performance metrics (power output, ascent velocity, speed), and workload and intensity measures. Differences in performance metrics and workload and intensity measures between groups were investigated. Power output, ascent velocity, speed were higher in professionals than U23 cyclists for Cat 1 and Cat 2 (p≤0.001-0.016). Workload and intensity measures (Worktotal, Worktotal∙km-1, Elevationgain, eTRIMP and eTRIMP∙km-1) were higher in U23 compared to professionals (p=0.002-0.014). Climbing performance metrics were significantly predicted by prior workload and intensity measures for Cat 1 and 2 (R2=0.27-0.89, p≤0.001-0.030) but not Cat 3. These findings reveal that climbing performance in professional road cycling is influenced by climb categorization as well as prior workload and intensity measures. Combined, these findings suggest that Cat 1 and 2 climbing performance could be predicted from workload and intensity measures.

Ergothioneine, Ovothiol A, and Selenoneine-Histidine-Derived, Biologically Significant, Trace Global Alkaloids.

The history, chemistry, biology, and biosynthesis of the globally occurring histidine-derived alkaloids ergothioneine (10), ovothiol A (11), and selenoneine (12) are reviewed comparatively and their significance to human well-being is discussed.

Detection and Verification of a Key Intermediate in an Enantioselective Peptide Catalyzed Acylation Reaction.

Until now, the intermediate responsible for the acyl transfer of a highly enantioselective tetrapeptide organocatalyst for the kinetic resolution of trans-cycloalkane-1,2-diols has never been directly observed. It was proposed computationally that a π-methylhistidine moiety is acylated as an intermediate step in the catalytic cycle. In this study we set out to investigate whether we can detect and characterize this key intermediate using NMR-spectroscopy and mass spectrometry. Different mass spectrometric experiments using a nano-ElectroSpray Ionization (ESI) source and tandem MS-techniques allowed the identification of tetrapeptide acylium ions using different acylation reagents. The complexes of trans-cyclohexane-1,2-diols with the tetrapeptide were also detected. Additionally, we were able to detect acylated tetrapeptides in solution using NMR-spectroscopy and monitor the acetylation reaction of a trans-cyclohexane-1,2-diol. These findings are important steps towards the understanding of this highly enantioselective organocatalyst.

Sulfur-containing histidine compounds inhibit γ-glutamyl transpeptidase activity in human cancer cells.

γ-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(Nπ)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent Ki of 21 µm, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-l-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy.

TMAO, creatine and 1-methylhistidine in serum and urine are potential biomarkers of cod and salmon intake: a randomised clinical trial in adults with overweight or obesity.

PURPOSE: To identify biomarkers to assess participants' compliance in an intervention study with high intake of cod or salmon, compared to a fish-free diet. METHODS: In this randomised clinical trial, 62 healthy overweight/obese participants consumed 750 g/week of either cod (N = 21) or salmon (N = 22) across 5 weekly dinners, or were instructed to continue their normal eating habits but avoid fish intake (Control group, N = 19) for 8 weeks. RESULTS: After cod intake, serum concentrations of trimethylamine N-oxide (TMAO, p = 0.0043), creatine (p = 0.024) and 1-methylhistidine (1-MeHis, p = 0.014), and urine concentrations (relative to creatinine) of TMAO (p = 2.8 × 10-5), creatine (p = 8.3 × 10-4) and 1-MeHis (p = 0.016) were increased when compared to Control group. After salmon intake, serum concentrations of 1-MeHis (p = 2.0 × 10-6) and creatine (p = 6.1 × 10-4), and urine concentrations (relative to creatinine) of 1-MeHis (p = 4.2 × 10-6) and creatine (p = 4.0 × 10-5) were increased when compared to Control group. Serum and urine concentrations of TMAO were more increased following cod intake compared to salmon intake (p = 0.028 and 2.9 × 10-4, respectively), and serum and urine 1-MeHis concentrations were more increased after salmon intake compared to cod intake (p = 8.7 × 10-5 and 1.2 × 10-4, respectively). Cod and salmon intake did not affect serum and urine concentrations of 3-methylhistidine, and only marginally affected concentrations of free amino acids and amino acid metabolites. CONCLUSION: TMAO measured in serum or urine is a potential biomarker of cod intake, and 1-MeHis measured in serum or urine is a potential biomarker of salmon intake.

Insights into the Light Response of Skeletonema marinoi: Involvement of Ovothiol.

Diatoms are one of the most widespread groups of microalgae on Earth. They possess extraordinary metabolic capabilities, including a great ability to adapt to different light conditions. Recently, we have discovered that the diatom Skeletonema marinoi produces the natural antioxidant ovothiol B, until then identified only in clams. In this study, we investigated the light-dependent modulation of ovothiol biosynthesis in S. marinoi. Diatoms were exposed to different light conditions, ranging from prolonged darkness to low or high light, also differing in the velocity of intensity increase (sinusoidal versus square-wave distribution). The expression of the gene encoding the key ovothiol biosynthetic enzyme, ovoA, was upregulated by high sinusoidal light mimicking natural conditions. Under this situation higher levels of reactive oxygen species and nitric oxide as well as ovothiol and glutathione increase were detected. No ovoA modulation was observed under prolonged darkness nor low sinusoidal light. Unnatural conditions such as continuous square-wave light induced a very high oxidative stress leading to a drop in cell growth, without enhancing ovoA gene expression. Only one of the inducible forms of nitric oxide synthase, nos2, was upregulated by light with consequent production of NO under sinusoidal light and darkness conditions. Our data suggest that ovothiol biosynthesis is triggered by a combined light stress caused by natural distribution and increased photon flux density, with no influence from the daily light dose. These results open new perspectives for the biotechnological production of ovothiols, which are receiving a great interest for their biological activities in human model systems.

Effects of rumen-protected lysine and histidine on milk production and energy and nitrogen utilization in diets containing hydrolyzed feather meal fed to lactating Jersey cows.

Hydrolyzed feather meal (HFM) is high in crude protein, most of which bypasses rumen degradation when fed to lactating dairy cows, allowing direct supply of AA to the small intestine. Compared with other feeds that are high in bypass protein, such as blood meal or heat-treated soybean meal, HFM is low in His and Lys. The objectives of this study were to determine the effects of supplementing rumen-protected (RP) Lys and His individually or in combination in a diet containing 5% HFM on milk production and composition as well as energy and N partitioning. Twelve multiparous Jersey cows (mean ± SD: 91 ± 18 d in milk) were used in a triplicated 4 × 4 Latin square with 4 periods of 28 d (24-d adaptation and 4-d collection). Throughout the experiment, all cows were fed the same TMR, with HFM included at 5% of diet DM. Cows were grouped by dry matter intake and milk yield, and cows within a group were randomly assigned to 1 of 4 treatments: no RP Lys or RP His; RP Lys only [70 g/d of Ajipro-L (24 g/d of digestible Lys), Ajinomoto Co. Inc., Tokyo, Japan]; RP His only [32 g/d of experimental product (7 g/d of digestible His), Balchem Corp., New Hampton, NY]; or both RP Lys and His. Plasma Lys concentration increased when RP Lys was supplemented without RP His (77.7 vs. 66.0 ± 4.69 µM) but decreased when RP Lys was supplemented with RP His (71.4 vs. 75.0 ± 4.69 µM). Plasma concentration of 3-methylhistidine decreased with RP Lys (3.19 vs. 3.40 ± 0.31 µM). With RP His, plasma concentration of His increased (21.8 vs. 18.7 ± 2.95 µM). For milk production and milk composition, no effects of Lys were observed. Supplementing RP His increased milk yield (22.5 vs. 21.6 ± 2.04 kg/d) and tended to increase milk protein yield (0.801 vs. 0.772 ± 0.051 kg/d). Across treatments, dry matter intake (18.5 ± 0.83 kg/d) and energy supply (32.2 ± 2.24 Mcal of net energy for lactation) were not different. Supplementing RP His did not affect N utilization; however, supplementing RP Lys increased N balance (25 vs. 16 ± 9 g/d). The lack of production responses to RP Lys suggests that Lys was not limiting or that the increase in Lys supply was not large enough to cause an increase in milk protein yield. However, increased N balance and decreased 3-methylhistidine with RP Lys suggest that increased Lys supply increased protein accretion and decreased protein mobilization. Furthermore, His may be a limiting AA in diets containing HFM.

Proteasome activity and expression of mammalian target of rapamycin signaling factors in skeletal muscle of dairy cows supplemented with conjugated linoleic acids during early lactation.

The mammalian target of rapamycin (mTOR) is a major regulator of protein synthesis via its main downstream effectors, ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein (4EBP1). The ubiquitin-proteasome system (UPS) is the main proteolytic pathway in muscle, and the muscle-specific ligases tripartite motif containing 63 (TRIM63; also called muscle-specific ring-finger protein 1, MuRF-1) and F-box only protein 32 (FBXO32; also called atrogin-1) are important components of the UPS. We investigated 20S proteasome activity and mRNA expression of key components of mTOR signaling and UPS in skeletal muscle of dairy cows during late gestation and early lactation and tested the effects of dietary supplementation (from d 1 in milk) with conjugated linoleic acids (sCLA; 100 g/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). Blood and muscle tissue (semitendinosus) samples were collected on d -21, 1, 21, and 70 relative to parturition. Dry matter intake increased with time of lactation in both groups. It was lower in sCLA than in CTR on d 21, which resulted in a reduced calculated metabolizable protein balance. Most serum and muscle concentrations of AA followed time-related changes but were unaffected by CLA supplementation. In both groups, serum and muscle 3-methylhistidine (3-MH) concentrations and the ratio of 3-MH:creatinine increased from d -21 to d 1, followed by a decline on d 21. The mRNA abundance of MTOR on d 21 and 70 was greater in sCLA than in CTR. The abundance of 4EBP1 mRNA did not differ between groups but was upregulated in both on d 1. The mRNA abundance of S6K1 on d 70 was greater in CTR than in sCLA, but remained unchanged over time in both groups. The mRNA abundance of FBXO32 (encoding atrogin-1) on d 21 was greater in sCLA than in CTR. The mRNA abundance of TRIM63 (also known as MuRF1) showed a similar pattern as FBXO32 in both groups: an increase from d -21 to d 1, followed by a decline. The mRNA for the α (BCKDHA) and ß (BCKDHB) polypeptide of branched-chain α-keto acid dehydrogenase was elevated in sCLA and CTR cows on d 21, respectively, suggesting a role of CLA in determining the metabolic fate of branched-chain AA. For the mTOR protein, no group differences were observed. The abundance of S6K1 protein was greater across all time points in sCLA versus CTR. The antepartum 20S proteasome activity in muscle was elevated in both groups compared with postpartum, probably reflecting the start of protein mobilization before parturition. Plasma insulin concentrations decreased in both groups postpartum but to a greater extent in CTR than in sCLA, resulting in greater insulin concentrations in sCLA than in CTR. Thus, the greater abundance of MTOR mRNA and S6K1 protein in sCLA compared with CTR might be mediated by the greater plasma insulin postpartum. The upregulation of MTOR mRNA in sCLA cows on d 21, despite greater FBXO32 mRNA abundance, may reflect a simultaneous activation of both anabolic and catabolic signaling pathways, likely resulting in greater protein turnover.

How Does Replacement of the Axial Histidine Ligand in Cytochrome c Peroxidase by Nδ-Methyl Histidine Affect Its Properties and Functions? A Computational Study.

Heme peroxidases have important functions in nature related to the detoxification of H2O2. They generally undergo a catalytic cycle where, in the first stage, the iron(III)-heme-H2O2 complex is converted into an iron(IV)-oxo-heme cation radical species called Compound I. Cytochrome c peroxidase Compound I has a unique electronic configuration among heme enzymes where a metal-based biradical is coupled to a protein radical on a nearby Trp residue. Recent work using the engineered Nδ-methyl histidine-ligated cytochrome c peroxidase highlighted changes in spectroscopic and catalytic properties upon axial ligand substitution. To understand the axial ligand effect on structure and reactivity of peroxidases and their axially Nδ-methyl histidine engineered forms, we did a computational study. We created active site cluster models of various sizes as mimics of horseradish peroxidase and cytochrome c peroxidase Compound I. Subsequently, we performed density functional theory studies on the structure and reactivity of these complexes with a model substrate (styrene). Thus, the work shows that the Nδ-methyl histidine group has little effect on the electronic configuration and structure of Compound I and little changes in bond lengths and the same orbital occupation is obtained. However, the Nδ-methyl histidine modification impacts electron transfer processes due to a change in the reduction potential and thereby influences reactivity patterns for oxygen atom transfer. As such, the substitution of the axial histidine by Nδ-methyl histidine in peroxidases slows down oxygen atom transfer to substrates and makes Compound I a weaker oxidant. These studies are in line with experimental work on Nδ-methyl histidine-ligated cytochrome c peroxidases and highlight how the hydrogen bonding network in the second coordination sphere has a major impact on the function and properties of the enzyme.

MicroRNA-140 inhibits skeletal muscle glycolysis and atrophy in endotoxin-induced sepsis in mice via the WNT signaling pathway.

Sepsis is a systemic inflammatory response syndrome resulting from infection. This study aimed at exploring the role of microRNA-140 (miR-140) in septic mice. Wnt family member 11 (WNT11) was verified to be a target gene of miR-140 after bioinformatic prediction and dual luciferase reporter gene assay. Importantly, miR-140 negatively regulated WNT11. We initially induced the model of sepsis by endotoxin, and then ectopic expression and knockdown experiments were performed to explore the functional role of miR-140 in sepsis. Additionally, cross-sectional areas of muscle fiber, lactic acid production, 3-methylhistidine (3-MH) and tyrosine (Tyr) production in extensor digitorium longus (EDL) muscles, and serum levels of inflammatory factors were examined. The effect of miR-140 on the expression of WNT signaling pathway-related and apoptosis-related factors in skeletal muscle tissue was determined. The experimental results indicated that upregulated miR-140 or silenced WNT11 increased cross-sectional areas of muscle fiber while decreasing lactic acid production, skeletal muscle cell apoptosis [corresponding to downregulated B cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and caspase-3 and upregulated Bcl-2], and the proteolytic rate of Tyr and 3-MH. Also, overexpressed miR-140 or silenced WNT11 reduced inflammation as reflected by decreased serum levels of IL-6, IL-10, and TNF-α. Furthermore, overexpression of miR-140 was shown to suppress the activation of the WNT signaling pathway, accompanied by decreased expression of WNT11, ß-catenin, and GSK-3ß. Taken together, upregulation of miR-140 could potentially inhibit skeletal muscle lactate release, an indirect measure of glycolysis, and atrophy in septic mice through suppressing the WNT signaling pathway via inhibiting WNT11 expression.

The complex evolutionary history of sulfoxide synthase in ovothiol biosynthesis.

Sulfoxide synthases are enzymes involved in the biosynthesis of small sulfur-containing natural products. Their enzymatic activity represents a unique sulfur transfer strategy in nature that is the insertion of a sulfur atom on the imidazole ring of histidine. To date, only two enzymes are known to carry out this function: the sulfoxide synthase EgtB, involved in the biosynthesis of ergothioneine in fungi and bacteria, and the 5-histidylcysteine sulfoxide synthase OvoA, involved in the biosynthesis of ovothiols, found in the eggs and biological fluids of marine invertebrates, some proteobacteria and protists. In particular, ovothiols, thanks to their unique redox properties, are probably the most intriguing marine sulfur-containing molecules. Although they have long been considered as cellular protective molecules, new evidence suggest that their biological activities and ecological role might be more complex than originally thought. Here, we investigate the evolutionary history of OvoA in Metazoa, reporting its monophyletic ancient origins, which could be traced back to the latest common ancestor of Choanozoa. Nevertheless, we show that OvoA is missing in several major extant taxa and we discuss this patchy distribution in the light of the massive genome reduction events documented in Metazoa. We also highlight two interesting cases of secondary acquisition through horizontal gene transfer, which occurred in hydrozoans and bdelloid rotifers. The evolutionary success of this metabolic pathway is probably ascribable to its role in the maintenance of cellular redox homeostasis, which enables organisms to survive in different environmental niches.

Mammalian target of rapamycin signaling and ubiquitin-proteasome-related gene expression in skeletal muscle of dairy cows with high or normal body condition score around calving.

The objective of the current study was to investigate the effects of overconditioning around calving on gene expression of key components of the mammalian target of rapamycin (mTOR) pathway and ubiquitin-proteasome system (UPS) in skeletal muscle as well as the AA profiles in both serum and muscle of periparturient cows. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to either a high body condition group (HBCS; n = 19) or a normal body condition group (NBCS; n = 19) and were fed different diets until dry-off (d -49 relative to calving) to amplify the difference. The groups were also stratified for comparable milk yields (NBCS: 10,361 ± 302 kg; HBCS: 10,315 ± 437 kg). At dry-off, the NBCS cows (parity: 2.42 ± 1.84; body weight: 665 ± 64 kg) had a body condition score (BCS) <3.5 and backfat thickness (BFT) <1.2 cm, whereas the HBCS cows (parity: 3.37 ± 1.67; body weight: 720 ± 57 kg) had a BCS >3.75 and BFT >1.4 cm. During the dry period and the subsequent lactation, both groups were fed identical diets but maintained the BCS and BFT differences. Blood samples and skeletal muscle biopsies (semitendinosus) were repeatedly (d -49, +3, +21, and +84 relative to calving) collected for assessing the concentrations of free AA and the mRNA abundance of various components of mTOR and UPS. The differences in BCS and BFT were maintained throughout the study. The circulating concentrations of most AA with the exception of Gly, Gln, Met, and Phe increased in early lactation in both groups. The serum concentrations of Ala (d +21 and +84) and Orn (d +84) were lower in HBCS cows than in NBCS cows, but those of Gly, His, Leu, Val, Lys, Met, and Orn on d -49 and Ile on d +21 were greater in HBCS cows than in NBCS cows. The serum concentrations of 3-methylhistidine, creatinine, and 3-methylhistidine:creatinine ratio increased after calving (d +3) but did not differ between the groups. The muscle concentrations of all AA (except for Cys) remained unchanged over time and did not differ between groups. The muscle concentrations of Cys were greater on d -49 but tended to be lower on d +21 in HBCS cows than in NBCS cows. On d +21, mTOR and eukaryotic translation initiation factor 4E binding protein 1 mRNA abundance was greater in HBCS cows than in NBCS cows, whereas ribosomal protein S6 kinase 1 was not different between the groups. The mRNA abundance of ubiquitin-activating enzyme 1 (d +21), ubiquitin-conjugating enzyme 1 (d +21), atrogin-1 (d +21), and ring finger protein-1 (d +3) enzymes was greater in HBCS cows than in NBCS cows, whereas ubiquitin-conjugating enzyme 2 was not different between the groups. The increased mRNA abundance of key components of mTOR signaling and of muscle-specific ligases of HBCS cows may indicate a simultaneous activation of anabolic and catabolic processes and thus increased muscle protein turnover, likely as a part of the adaptive response to prevent excessive loss of skeletal muscle mass during early lactation.