Search Results (28)

Vitamin E deficiency (VED) represents a common micronutrient deficiency in dairy cows (DCs), leading to severe degenerative diseases, oxidative stress, immune dysfunction, and various health issues, ultimately causing significant economic losses for the global dairy sector. Accordingly, our objective was to explore the metabolic features of VED-afflicted cows by combining the untargeted gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) and targeted liquid chromatography-mass spectrometry (LC-MS) to identify effective serum VED biomarkers. Untargeted GC-TOF-MS analysis identified 31 differential metabolites (DMs): 20 were overexpressed and 11 were suppressed in the VED group compared to the healthy control group. These DMs were enriched in six major metabolic pathways: glycine, serine, and threonine; alanine, aspartate, and glutamate; cysteine and methionine; tyrosine; primary bile acid biosynthesis; and nitrogen metabolisms. These outcomes show that VED significantly disrupts amino acid/lipid/energy metabolism pathways in DCs. Further targeted LC-MS quantification revealed significant alterations in key metabolites, including increased levels of norepinephrine, glycine, cysteine, and L-glutamine, as well as a significant reduction in cholesterol concentrations. Binary logistic regression analysis identified norepinephrine and cholesterol as strong candidate biomarkers for VED. Receiver operating characteristic curve analysis established outstanding diagnostic accuracy for norepinephrine and cholesterol (for both p < 0.001, area under the curve = 0.980 and 0.990, correspondingly), with sensitivities and specificities of 90% and 100%, respectively. In conclusion, this study integrates untargeted and targeted metabolomics approaches to reveal VED-caused metabolic disruptions in DCs, particularly in amino acid/lipid/energy metabolism pathways. Norepinephrine and cholesterol were identified as highly accurate serum VED biomarkers with excellent diagnostic performance. Early detection and timely intervention using these biomarkers could promote disease treatment and cow health, as well as productivity, and decrease economic losses. Full article

Nattokinase (NK), a serine protease with high thrombolytic activity, has significant potential for application in foods intended for special health benefits. However, the NK production in wild-type Bacillus subtilis natto is relatively low. In this study, a high-yielding NK and genetically stable mutant strain (B. subtilis JNC002.001, 300.0 ± 4.7 FU/mL) was obtained through atmospheric and room temperature plasma (ARTP) mutagenesis. It increased NK activity by 1.84 times compared to the initial strain SD2, demonstrating significant prospects for NK production and food fermentation applications. Additionally, the B. subtilis JNC002.001 exhibited notable alterations in growth characteristics, glucose consumption, and sporulation. This study further elucidated the mechanism of enhanced NK production at the molecular level. Genome resequencing revealed that the mutant genes in JNC002.001 included 10 single nucleotide polymorphisms (SNPs) and one insertion, among which the kinA and gltA genes were associated with sporulation and NK synthesis, respectively. In terms of the transcriptional level, the NK-coding gene aprN was up-regulated 9.4 times relative to the wild-type strain. Most of the genes related to central carbon metabolism and the Sec secretion pathway were up-regulated. In addition, the expression of regulatory factors associated with the transcription of the aprN gene and the sporulation process provided evidence for high NK expression and sporulation deficiency in JNC002.001. These results could provide insights into the mechanism of NK production and facilitate the construction of engineered strains with high NK yield. Full article

Background: Elongation factor protein (EF-P) in bacteria helps ribosomes to incorporate contiguous proline residues (xPro) into proteins. In this way, EF-P rescues ribosomes from stalling at these xPro motifs. Whereas EF-P deficiency is lethal for some species, others show reduced virulence or generally lower growth rates, such as Escherichia coli (E. coli). EF-P needs to be post-translationally modified to gain full functionality. Methods: We constructed E. coli K-12 mutant strains with deletion of the serA gene leading to an auxotrophy for L-serine. Then, we engineered a 6xPro motif in the recombinant serA gene, which was then chromosomally inserted under its native promoter. Furthermore, mutant strains which were deleted for efp and/or epmA (encoding the EF-P modification protein EpmA) were engineered. Results: Δefp, ΔepmA, and Δefp/ΔepmA double mutants showed already significantly reduced growth rates in minimal media. ΔserA derivatives of these strains were complemented by the wt serA gene but not by 6xPro-serA. ΔserA mutants with intact efp were complemented by all serA-constructs. Chromosomal expression of the recombinant efp gene from E. coli or from the pathogen, Staphylococcus aureus (S. aureus), restored growth, even without epmA expression. Conclusions: We provide a novel synthetic reporter system for in vivo evaluation of EF-P deficiency. In addition, we demonstrated that both EF-P-E. coli and EF-P-S. aureus restored the growth of a 6xPro-serA: Δefp, ΔepmA strain, which is evidence that modification of EF-P might be dispensable for rescuing of ribosomes stalled during translation of proline repeats. Full article

In humans, the phosphorylated pathway (PP) converts the glycolytic intermediate D-3-phosphoglycerate (3-PG) into L-serine through the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase. From the pathogenic point of view, the PP in the brain is particularly relevant, as genetic defects of any of the three enzymes are associated with a group of neurometabolic disorders known as serine deficiency disorders (SDDs). We recombinantly expressed and characterized eight variants of PSAT associated with SDDs and two non-SDD associated variants. We show that the pathogenetic mechanisms in SDDs are extremely diverse, including low affinity of the cofactor pyridoxal 5′-phosphate and thermal instability for S179L and G79W PSAT, loss of activity of the holo form for R342W PSAT, aggregation for D100A PSAT, increased K_m for one of the substrates with invariant k_cats for S43R PSAT, and a combination of increased K_m and decreased k_cat for C245R PSAT. Finally, we show that the flux through the in vitro reconstructed PP at physiological concentrations of substrates and enzymes is extremely sensitive to alterations of the functional properties of PSAT variants, confirming PSAT dysfunctions as a cause of SSDs. Full article

L-serine is a non-essential amino acid that plays a vital role in protein synthesis, cell proliferation, development, and sphingolipid formation in the central nervous system. It exerts its effects through the activation of glycine receptors and upregulation of PPAR-γ, resulting in neurotransmitter synthesis, neuroprotection, and anti-inflammatory effects. L-serine shows potential as a protective agent in various neurological diseases and neurodegenerative disorders. Deficiency of L-serine and its downstream products has been linked to severe neurological deficits. Despite its crucial role, there is limited understanding of its mechanistic production and impact on glial and neuronal cells. Most of the focus has been on D-serine, the downstream product of L-serine, which has been implicated in a wide range of neurological diseases. However, L-serine is approved by FDA for supplemental use, while D-serine is not. Hence, it is imperative that we investigate the wider effects of L-serine, particularly in relation to the pathogenesis of several neurological deficits that, in turn, lead to diseases. This review aims to explore current knowledge surrounding L-serine and its potential as a treatment for various neurological diseases and neurodegenerative disorders. Full article

Background: Epidemiologic studies have reported that the geographical distribution of the prevalence of allelic variants of serine protein inhibitor-A1 (SERPINA1) and severe cases of COVID-19 were similar. Methods: A multicenter, cross-sectional, observational study to evaluate the frequency of alpha-1 antitrypsin deficiency (AATD) in patients with COVID-19 and whether it was associated with having suffered severe COVID-19. Results: 2022 patients who had laboratory-confirmed SARS-CoV-2 infection. Mutations associated with AATD were more frequent in severe COVID versus non-severe (23% vs. 18.8%, p = 0.022). The frequency of Pi*Z was 37.8/1000 in severe COVID versus 17.5/1000 in non-severe, p = 0.001. Having an A1AT level below 116 was more frequent in severe COVID versus non-severe (29.5% vs. 23.1, p = 0.003). Factors associated with a higher likelihood of severe COVID-19 were being male, older, smoking, age-associated comorbidities, and having an A1AT level below 116 mg/dL [OR 1.398, p = 0.003], and a variant of the SERPINA1 gene that could affect A1AT protein [OR 1.294, p = 0.022]. Conclusions: These observations suggest that patients with AATD should be considered at a higher risk of developing severe COVID-19. Further studies are needed on the role of A1AT in the prognosis of SARS-CoV-2 infection and its possible therapeutic role. Full article

The most frequent alterations in plasma amino acid concentrations in type 1 and type 2 diabetes are decreased L-serine and increased branched-chain amino acid (BCAA; valine, leucine, and isoleucine) levels. The likely cause of L-serine deficiency is decreased synthesis of 3-phosphoglycerate, the main endogenous precursor of L-serine, due to impaired glycolysis. The BCAA levels increase due to decreased supply of pyruvate and oxaloacetate from glycolysis, enhanced supply of NADH + H⁺ from beta-oxidation, and subsequent decrease in the flux through the citric acid cycle in muscles. These alterations decrease the supply of α-ketoglutarate for BCAA transamination and the activity of branched-chain keto acid dehydrogenase, the rate-limiting enzyme in BCAA catabolism. L-serine deficiency contributes to decreased synthesis of phospholipids and increased synthesis of deoxysphinganines, which play a role in diabetic neuropathy, impaired homocysteine disposal, and glycine deficiency. Enhanced BCAA levels contribute to increased levels of aromatic amino acids (phenylalanine, tyrosine, and tryptophan), insulin resistance, and accumulation of various metabolites, whose influence on diabetes progression is not clear. It is concluded that amino acid concentrations should be monitored in patients with diabetes, and systematic investigation is needed to examine the effects of L-serine and glycine supplementation on diabetes progression when these amino acids are decreased. Full article

Neurodegenerative diseases feature changes in cognition, and anxiety-like and autism-like behaviors, which are associated with epigenetic alterations such as DNA methylation and histone modifications. The amino acid L-serine has been shown to have beneficial effects on neurological symptoms. Here, we found that growth hormone-releasing hormone knockout (GHRH-KO) mice, a GH-deficiency mouse model characterized by extended lifespan and enhanced insulin sensitivity, showed a lower anxiety symptom and impairment of short-term object recognition memory and autism-like behaviors. Interestingly, L-serine administration exerted anxiolytic effects in mice and ameliorated the behavioral deficits in GHRH-KO. L-serine treatment upregulated histone epigenetic markers of H3K4me, H3K9ac, H3K14ac and H3K18ac in the hippocampus and H3K4me in the cerebral cortex in both GHRH-KO mice and wild type controls. L-serine-modulated epigenetic marker changes, in turn, were found to regulate mRNA expression of BDNF, grm3, foxp1, shank3, auts2 and marcksl1, which are involved in anxiety-, cognitive- and autism-like behaviors. Our study provides a novel insight into the beneficial effects of L-serine intervention on neuropsychological impairments. Full article

Protein homeostasis, including protein folding, refolding, and degradation, is thought to decline with aging. HSPB5 (also known as αB-crystallin) prevents target protein aggregation as a molecular chaperone and exhibits a cytoprotective function against various cell stresses. To elucidate the effect of HSPB5 on endoplasmic reticulum (ER) stress, we searched for novel binding proteins of HSPB5 using the proximity-dependent biotin labeling method. Proteins presumed to interact with HSPB5 in cells treated with the proteasome inhibitor MG132 were identified by a reversible biotin-binding capacity method combining tamavidin2-REV magnetic beads and mass spectrometry. We discovered a new binding protein for HSPB5, polo-like kinase 2 (PLK2), which is an apoptosis-related enzyme. The expression of PLK2 was upregulated by MG132 treatment, and it was co-localized with HSPB5 near the ER in L6 muscle cells. Inhibition of PLK2 decreased ER stress-induced phosphorylation of serine 19 in HSPB5 and increased apoptosis by activation of caspase 3 under ER stress. Overexpression of HSPB5 (WT) suppressed the ER stress-induced caspase 3 activity, but this was not observed with phospho-deficient HSPB5 (3A) mutants. These results clarify the role of HSPB5 phosphorylation during ER stress and suggest that the PLK2/HSPB5 pathway plays an essential role in cytoprotection against proteasome inhibition-induced ER stress. Full article

L-serine plays an essential role in a broad range of cellular functions including protein synthesis, neurotransmission, and folate and methionine cycles and synthesis of sphingolipids, phospholipids, and sulphur containing amino acids. A hydroxyl side-chain of L-serine contributes to polarity of proteins, and serves as a primary site for binding a phosphate group to regulate protein function. D-serine, its D-isoform, has a unique role. Recent studies indicate increased requirements for L-serine and its potential therapeutic use in some diseases. L-serine deficiency is associated with impaired function of the nervous system, primarily due to abnormal metabolism of phospholipids and sphingolipids, particularly increased synthesis of deoxysphingolipids. Therapeutic benefits of L-serine have been reported in primary disorders of serine metabolism, diabetic neuropathy, hyperhomocysteinemia, and amyotrophic lateral sclerosis. Use of L-serine and its metabolic products, specifically D-serine and phosphatidylserine, has been investigated for the therapy of renal diseases, central nervous system injury, and in a wide range of neurological and psychiatric disorders. It is concluded that there are disorders in which humans cannot synthesize L-serine in sufficient quantities, that L-serine is effective in therapy of disorders associated with its deficiency, and that L-serine should be classified as a “conditionally essential” amino acid. Full article

Inhibition of proteases shows therapeutic potential. Our previous studies demonstrated the cardioprotection by the Secretory Leukocyte Protease Inhibitor (SLPI) against myocardial ischaemia/reperfusion (I/R) injury. However, it is unclear whether the cardioprotective effect of SLPI seen in our previous works is due to the inhibition of protease enzymes. Several studies demonstrate that the anti-protease independent activity of SLPI could provide therapeutic benefits. Here, we show for the first time that recombinant protein of anti-protease deficient mutant SLPI (L72K, M73G, L74G) (mt-SLPI) could significantly reduce cell death and intracellular reactive oxygen species (ROS) production against an in vitro simulated I/R injury. Furthermore, post-ischaemic treatment of mt-SLPI is found to significantly reduce infarct size and cardiac biomarkers lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity, improve cardiac functions, attenuate I/R induced-p38 MAPK phosphorylation, and reduce apoptotic regulatory protein levels, including Bax, cleaved-Caspase-3 and total Capase-8, in rats subjected to an in vivo I/R injury. Additionally, the beneficial effect of mt-SLPI was not significantly different from the wildtype (wt-SLPI). In summary, SLPI could provide cardioprotection without anti-protease activity, which could be more clinically beneficial in terms of providing cardioprotection without interfering with basal serine protease activity. Full article

Pigeon Pea (Cajanus cajan (L.) Millsp.) is a common food crop used in many parts of the world for nutritional purposes. One of its chemical constituents is cajanin stilbene acid (CSA), which exerts anticancer activity in vitro and in vivo. In an effort to identify molecular targets of CSA, we performed a kinome-wide approach based on the measurement of the enzymatic activities of 252 human kinases. The serine-threonine kinase WNK3 (also known as protein kinase lysine-deficient 3) was identified as the most promising target of CSA with the strongest enzymatic activity inhibition in vitro and the highest binding affinity in molecular docking in silico. The lowest binding affinity and the predicted binding constant pKi of CSA (−9.65 kcal/mol and 0.084 µM) were comparable or even better than those of the known WNK3 inhibitor PP-121 (−9.42 kcal/mol and 0.123 µM). The statistically significant association between WNK3 mRNA expression and cellular responsiveness to several clinically established anticancer drugs in a panel of 60 tumor cell lines and the prognostic value of WNK3 mRNA expression in sarcoma biopsies for the survival time of 230 patients can be taken as clues that CSA-based inhibition of WNK3 may improve treatment outcomes of cancer patients and that CSA may serve as a valuable supplement to the currently used combination therapy protocols in oncology. Full article

l-Serine (Ser) is synthesized de novo from 3-phosphoglycerate via the phosphorylated pathway committed by phosphoglycerate dehydrogenase (Phgdh). A previous study reported that feeding a protein-free diet increased the enzymatic activity of Phgdh in the liver and enhanced Ser synthesis in the rat liver. However, the nutritional and physiological functions of Ser synthesis in the liver remain unclear. To clarify the physiological significance of de novo Ser synthesis in the liver, we generated liver hepatocyte-specific Phgdh KO (LKO) mice using an albumin-Cre driver. The LKO mice exhibited a significant gain in body weight compared to Floxed controls at 23 weeks of age and impaired systemic glucose metabolism, which was accompanied by diminished insulin/IGF signaling. Although LKO mice had no apparent defects in steatosis, the molecular signatures of inflammation and stress responses were evident in the liver of LKO mice. Moreover, LKO mice were more vulnerable to protein starvation than the Floxed mice. These observations demonstrate that Phgdh-dependent de novo Ser synthesis in liver hepatocytes contributes to the maintenance of systemic glucose tolerance, suppression of inflammatory response, and resistance to protein starvation. Full article

Glutaric aciduria type II (GA-II) is a rare autosomal recessive disease caused by defects in electron transfer flavoprotein (ETF), ultimately causing insufficiencies in multiple acyl-CoA dehydrogenase (MAD). 3-phosphoglycerate dehydrogenase (3-PHGDH) deficiency, is another rare autosomal disorder that appears due to a defect in the synthesis of L-serine amino acid. Several mutations of ETFDH and PHGDH genes have been associated with different forms of GA-II and serine deficiency, respectively. In this study, we report a unique case of GA-II with serine deficiency using biochemical, genetic, and in silico approaches. The proband of Syrian descent had positive newborn screening (NBS) for GA-II. At two years of age, the patient presented with developmental regression, ataxia, and intractable seizures. Results of amino acid profiling demonstrated extremely low levels of serine. Confirmatory tests for GA-II and whole exome sequencing (WES) were performed to determine the etiology of intractable seizure. Sequencing results indicated a previously reported homozygous missense mutation, c.679 C>A (p.Pro227Thr) in the ETFDH gene and a novel missense homozygous mutation c.1219 T>C (p.Ser407Pro) in the PHGDH gene. In silico tools predicted these mutations as deleterious. Here, the clinical and biochemical investigations indicate that ETFDH:p.Pro227Thr and PHGDH:p.Ser407Pro variants likely underlie the pathogenesis of GA-II and serine deficiency, respectively. This study indicates that two rare autosomal recessive disorders should be considered in consanguineous families, more specifically in those with atypical presentation. Full article

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD⁺/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders. Full article