Protocol for mass spectrometric profiling of lysine malonylation by lysine acetyltransferase in CRISPRi K562 cell lines.

Lysine malonylation is a protein posttranslational modification. We present a protocol to generate stable gene-knockdown K562 cell lines through lentiviral infection of a CRISPR interference (CRISPRi) system followed by lysine malonylation measurement using mass spectrometry (MS). We detail guide RNA (gRNA) vector cloning, lentiviral infection, cell line purification, protein digestion, malonyl-lysine enrichment, desalting, and MS acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Zhang et al.1 and Bons et al.2.

A model of mitochondrial superoxide production during ischaemia-reperfusion injury for therapeutic development and mechanistic understanding.

Ischaemia-reperfusion (IR) injury is the paradoxical consequence of the rapid restoration of blood flow to an ischaemic organ. Although reperfusion is essential for tissue survival in conditions such as myocardial infarction and stroke, the excessive production of mitochondrial reactive oxygen species (ROS) upon reperfusion initiates the oxidative damage that underlies IR injury, by causing cell death and inflammation. This ROS production is caused by an accumulation of the mitochondrial metabolite succinate during ischaemia, followed by its rapid oxidation upon reperfusion by succinate dehydrogenase (SDH), driving superoxide production at complex I by reverse electron transport. Inhibitors of SDH, such as malonate, show therapeutic potential by decreasing succinate oxidation and superoxide production upon reperfusion. To better understand the mechanism of mitochondrial ROS production upon reperfusion and to assess potential therapies, we set up an in vitro model of IR injury. For this, isolated mitochondria were incubated anoxically with succinate to mimic ischaemia and then rapidly reoxygenated to replicate reperfusion, driving a burst of ROS formation. Using this system, we assess the factors that contribute to the magnitude of mitochondrial ROS production in heart, brain, and kidney mitochondria, as well as screening for inhibitors of succinate oxidation with therapeutic potential.

ROS-Suppression Nanoplatform Combined Activation of STAT3/Bcl-2 Pathway for Preventing Myocardial Infarction in Mice.

Myocardial infarction (MI) is the leading cause of death worldwide. The most effective way to treat myocardial infarction is to rescue ischemic cardiomyocytes. After an ischemic event, the overproduction of reactive oxygen species (ROS) is a key driver of myocardial injury. The produced ROS affects mitochondrial function and induces apoptosis in cardiomyocytes. This was accomplished by constructing platelet-membrane-encapsulated ROS-responsive drug-releasing nanoparticles (PMN@NIC-MalNPs) to deliver malonate and niclosamide (NIC). The results revealed that PMN@NIC-MalNPs degraded and released malonate and niclosamide in a high-level ROS microenvironment, effectively reducing the oxidative stress and apoptosis rate. By enhancing basal mitochondrial oxygen consumption rate (OCR), adenosine triphosphate (ATP) production, and spare respiratory capacity (SRC) in vitro, reduced the oxidative stress levels and restored mitochondrial function. In vivo studies revealed that the PMN@NIC-MalNPs improved cardiac dysfunction, inhibited succinate dehydrogenase (SDH) activity, increased ATP production, and reduced the myocardial infarct size in myocardial infarction model mice. Further, transcriptome analysis and Western blot revealed that PMN@NIC-MalNPs prevented apoptosis by activating the expressions of the signal transducer and activator of transcription 3 (STAT3) and Bcl-2, and inhibiting the expression of Bax. Thus, this study provides a novel therapeutic solution for treating myocardial infarction and predicting the viability of an antioxidant and antiapoptotic therapeutic solution in the treatment of myocardial injury.

Metabolomics analysis of human cumulus cells obtained from cumulus-oocyte complexes with different developmental potential.

STUDY QUESTION: Is the abundance of certain biochemical compounds in human cumulus cells (CCs) related to oocyte quality? SUMMARY ANSWER: Malonate, 5-oxyproline, and erythronate were positively associated with pregnancy potential. WHAT IS KNOWN ALREADY: CCs are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Mitochondrial DNA content and transcriptional analyses in CC have been shown to provide a poor predictive value of oocyte competence, but the untargeted analysis of biochemical compounds (metabolomics) has been unexplored. STUDY DESIGN, SIZE, DURATION: CCs were obtained from three groups of cumulus-oocyte complexes (COCs) of known developmental potential: oocytes not developing to blastocyst following ICSI (Bl-); oocytes developing to blastocyst but failing to establish pregnancy following embryo transfer (P-); and oocytes developing to blastocyst able to establish a pregnancy (P+). Metabolomics analyses were performed on 12 samples per group, each sample comprising the CC recovered from a single COC. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human CC samples were obtained from IVF treatments. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Metabolomics analysis was performed by ultra-high performance liquid chromatography-tandem mass spectroscopy. MAIN RESULTS AND THE ROLE OF CHANCE: The analysis identified 98 compounds, five of which were differentially abundant (P < 0.05) between groups: asparagine, proline, and malonate were less abundant in P- compared to Bl-, malonate and 5-oxoproline were less abundant in P- group compared to P+, and erythronate was less abundant in Bl- group compared to P+. No significant association between the abundance of the compounds identified and donor age or BMI was noted. LIMITATIONS, REASONS FOR CAUTION: Data dispersion and the lack of coherence between developmental groups preclude the direct use of metabolic markers in clinical practice, where the uterine environment plays a major role in pregnancy outcome. The abundance of other compounds not detected by the analysis may be associated with oocyte competence. As donors were lean (only two with BMI > 30 kg/m2) and young (<34 years old), a possible effect of obesity or advanced age on the CC metabolome could not be determined. WIDER IMPLICATIONS OF THE FINDINGS: The abundance of malonate, 5-oxyproline, and erythronate in CC was significantly higher in COCs ultimately establishing pregnancy, providing clues on the pathways required for oocyte competence. The untargeted analysis uncovered the presence of compounds that were not expected in CC, such as ß-citrylglutamate and the neurotransmitter N-acetyl-aspartyl-glutamate, which may play roles in chromatin remodeling and signaling, respectively. STUDY FUNDING/COMPETING INTEREST(S): Research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by Madrid Region Government. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

Synthesis, Characterization, and Bioactivity of the Lichen Pigments Pulvinamide, Rhizocarpic Acid, and Epanorin and Congeners.

The lichen natural products pulvinamide, rhizocarpic acid, and epanorin have been synthesized and characterized spectroscopically and by X-ray crystallography. The syntheses, by ring-opening of pulvinic acid dilactone (PAD), may well be biomimetic, given the well-known occurrence of PAD in lichen. The enantiomers, ent-rhizocarpic acid and ent-epanorin, and corresponding carboxylic acids, norrhizocarpic acid and norepanorin, were similarly prepared. All compounds were assessed for growth inhibitory activity against selected bacteria, fungi, a protist, a mammalian tumor cell line, and normal cells. Rhizocarpic acid is weakly antibacterial (Bacillus subtilis MIC = 50 µg/mL) and possesses modest but selective antitumor activity (NS-1 murine myeloma MIC = 3.1 µg/mL) with >10-fold potency relative to its enantiomer (MIC = 50 µg/mL).

Engineering a Novel Metabolic Pathway for Improving Cellular Malonyl-CoA Levels in Escherichia coli.

Cellular pool of malonyl-CoA in Escherichia coli is small, which impedes its utility for overproduction of natural products such as phenylpropanoids, polyketides, and flavonoids. In this study, we report the use of a new metabolic pathway to increase the malonyl-CoA concentration as a limiting metabolite in E. coli. For this purpose, the malonate/sodium symporter from Malonomonas rubra, and malonyl-CoA synthetase (MCS) from Bradyrhizobium japonicum were co-expressed in E. coli. This new pathway allows the cell to actively import malonate from the culture medium and to convert malonate and CoA to malonyl-CoA via an ATP-dependent ligation reaction. HPLC analysis confirmed elevated levels of malonyl-CoA and (2S)-naringenin as a malonyl-CoA-dependent metabolite, in E. coli. A 6.8-fold and more than 3.5-fold increase in (2S)-naringenin production were achieved in the engineered host in comparison with non-engineered E. coli and previously reported passive transport MatBMatC pathway, respectively. This observation suggests that using active transporters of malonate not only improves malonyl-CoA-dependent production but also makes it possible to harness low concentrations of malonate in culture media.

Systematic analysis of lysine malonylation in Streptococcus mutans.

Protein lysine malonylation (Kmal) is a novel post-translational modification (PTM) that regulates various biological pathways such as energy metabolism and translation. Malonylation in prokaryotes, however, is still poorly understood. In this study, we performed a global Kmal analysis of the cariogenic organism Streptococcus mutans by combining antibody-based affinity enrichment and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. Altogether, 392 malonyllysine sites in 159 proteins were identified. Subsequent bioinformatic analysis revealed that Kmal occurs in proteins involved in various metabolic pathways including translation machinery, energy metabolism, RNA degradation, and biosynthesis of various secondary metabolites. Quantitative analysis demonstrated that Kmal substrates were globally altered in the biofilm growth state compared to the planktonic growth state. Furthermore, a comparative analysis of the lysine malonylome of our study with previously determined lysine acetylome in S. mutans revealed that a small proportion of Kmal sites overlapped with acetylated sites, whereby suggesting that these two acylations have distinct functional implications. These results expand our knowledge of Kmal in prokaryotes, providing a resource for researching metabolic regulation of bacterial virulence and physiological functions by PTM.

Proteome-wide analyses reveal diverse functions of protein acetylation and succinylation modifications in fast growing stolons of bermudagrass (Cynodon dactylon L.).

BACKGROUND: Bermudagrass (Cynodon dactylon L.) is an important warm-season turfgrass species with well-developed stolons, which lay the foundation for the fast propagation of bermudagrass plants through asexual clonal growth. However, the growth and development of bermudagrass stolons are still poorly understood at the molecular level. RESULTS: In this study, we comprehensively analyzed the acetylation and succinylation modifications of proteins in fast-growing stolons of the bermudagrass cultivar Yangjiang. A total of 4657 lysine acetylation sites on 1914 proteins and 226 lysine succinylation sites on 128 proteins were successfully identified using liquid chromatography coupled to tandem mass spectrometry, respectively. Furthermore, 78 proteins and 81 lysine sites were found to be both acetylated and succinylated. Functional enrichment analysis revealed that acetylated proteins regulate diverse reactions of carbohydrate metabolism and protein turnover, whereas succinylated proteins mainly regulate the citrate cycle. These results partly explained the different growth disturbances of bermudagrass stolons under treatment with sodium butyrate and sodium malonate, which interfere with protein acetylation and succinylation, respectively. Moreover, 140 acetylated proteins and 42 succinylated proteins were further characterized having similarly modified orthologs in other grass species. Site-specific mutations combined with enzymatic activity assays indicated that the conserved acetylation of catalase and succinylation of malate dehydrogenase both inhibited their activities, further implying important regulatory roles of the two modifications. CONCLUSION: In summary, our study implied that lysine acetylation and succinylation of proteins possibly play important regulatory roles in the fast growth of bermudagrass stolons. The results not only provide new insights into clonal growth of bermudagrass but also offer a rich resource for functional analyses of protein lysine acetylation and succinylation in plants.

Global profiling of protein lysine malonylation in mouse cardiac hypertrophy.

Lysine malonylation, a novel identified protein posttranslational modification (PTM), is conservative and present in both eukaryotic and prokaryotic cells. Previous studies have reported that malonylation plays an important role in inflammation, angiogenesis, and diabetes. However, its potential role in cardiac remodeling remains unknown. Here, we observed a reduced lysine malonylation in hypertrophic mice hearts created by transverse aortic constriction (TAC) for 8 weeks. We also detected a decreased lysine malonylation in hypertrophic H9C2 cardiomyocytes induced by angiotensin II for 48 h. Using a proteomic method based on affinity purification and LC-MS/MS, we identified total 679 malonylated sites in 330 proteins in the hearts of sham mice and TAC mice. Bioinformatic analysis of the proteomic data revealed enrichment of malonylated proteins involved in cardiac structure and contraction, cGMP-PKG pathway, and metabolism. Specifically, we detected a decreased lysine malonylation in myocardial isocitrate dehydrogenase 2 (IDH2) by immunoprecipitation coupled with Western blotting both in vivo and in vitro. Together, our work suggests an important role and implication of protein lysine malonylation in cardiac hypertrophy, especially the IDH2. SIGNIFICANCE: Heart failure is the terminal stage of cardiac hypertrophy, which imposes an enormous clinical and economic burden worldwide. Despite our knowledge on the pathophysiology of the disease, current therapeutic approaches are still largely limited. Cardiac hypertrophy can be regulated at post-translational modifications (PTMs), and several PTMs have been reported in cardiac hypertrrophy and heart failure. In our study, we first reported a novel PTMs, lysine malonylation, in cardiac hypertophy. we found a reduced lysine malonylation in hypertrophic mice hearts in vivo and H9C2 cardiomyocytes after stimulating with angiotensinII for 48 h in vitro. Using affinity purification and LC-MS/MS, we identified 679 malonylated sites in 330 proteins in the hearts of sham and TAC mice. Compared to the sham group, 5 sites in 2 proteins were quantified as downregulated targets using a 2-fold threshold (downregulation <0.5-fold, P < 0.05). Functional analysis showed a significant enrichment in cardiac structure and contraction, cGMP-PKG pathway and metabolism. Notably, we identified a decreased Kmal level in isocitrate dehydrogenase 2 (IDH2), but the protein level of IDH2 has no changed in cardiac hypertrophy, These results highlight that lysine malonylation is associated with cardiac hypertrophy, and may be a new therapeutic target of the disease.

[Metabolic engineering of Escherichia coli for production of malonic acid].

Malonic acid is an important dicarboxylic acid, which can be widely used in the fields of chemical industry, medicine and food. In this study, a recombinant Escherichia coli strain BL21(TPP) was constructed to synthesize malonate through overexpressing six genes of ppc, aspC, panD, pa0132, yneI and pyc. Under shake flask fermentation conditons, strain BL21(TPP) produced 0.61 g/L malonic acid. In a 5 L fermentor, the production of malonic acid reached 3.32 g/L by using an intermittent feeding strategy. Next, a recombinant strain BL21(SCR) was constructed by fusional expression of ppc and aspC, as well as pa0132 and yneI, respectively. As a result, the production of malonic acid increased to 0.83 g/L at the shake flask level, which was a 36% increase over the starting strain BL21(TPP). Finally, the highest malonate production reached 5.61 g/L in a 5 L fermentor, which was a 69% increase over the starting strain BL21(TPP). Production of malonic acid by metabolically engineered E. coli provides a basis for further optimization, and may also serve as a reference for the biosynthesis of other dicarboxylic acids.

Metabolic engineering of the malonyl-CoA pathway to efficiently produce malonate in Saccharomyces cerevisiae.

Malonate is a platform chemical that has been utilized to synthesize many valuable chemical compounds. Here, Saccharomyces cerevisiae was metabolically engineered to produce malonate through the malonyl-CoA pathway. To construct the key step of converting malonyl-CoA to malonate, a native mitochondrial 3-hydroxyisobutyryl-CoA hydrolase gene EHD3 was mutated to target the cytoplasm and obtain malonyl-CoA hydrolase activity. The malonyl-CoA hydrolase activity of Ehd3 was achieved by mutating the malonyl-CoA binding site F121 to I121 and the active site E124 to seven amino acids (S/T/H/K/R/N/Q). We identified that the strain with E124S mutation had the highest malonate titer with 13.6 mg/L. Genomic integration of the mutant EHD3 and ACC1** to delta sequence sites was further explored to increase their reliable expression. Accordingly, a screening method with the work flow of fluorescence detection, shake-tube fermentation, and shake-flask fermentation was constructed to screen high copy delta sequences efficiently. The malonate titer was improved to 73.55 mg/L after screening the ∼1500 integrative strains, which was increased 4.4-folds than that of the episomal strain. We further engineered the strain by regulating the expression of key enzyme in the malonyl-CoA pathway to improve the precursor supply and inhibiting its competing pathways, and the final engineered strain LMA-16 produced 187.25 mg/L in the flask, 14-fold compared with the initial episomal expression strain. Finally, the combined efforts increased the malonate titer to 1.62 g/L in fed-batch fermentation.

Loss of the mitochondrial phosphate carrier SLC25A3 induces remodeling of the cardiac mitochondrial protein acylome.

Mitochondria are recognized as signaling organelles, because under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased posttranslational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel cross talk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function.

Post-Translational Formation of Aminomalonate by a Promiscuous Peptide-Modifying Radical SAM Enzyme.

Aminomalonate (Ama) is a widespread structural motif in Nature, whereas its biosynthetic route is only partially understood. In this study, we show that a radical S-adenosylmethionine (rSAM) enzyme involved in cyclophane biosynthesis exhibits remarkable catalytic promiscuity. This enzyme, named three-residue cyclophane forming enzyme (3-CyFE), mainly produces cyclophane in vivo, whereas it produces formylglycine (FGly) as a major product and barely produce cyclophane in vitro. Importantly, the enzyme can further oxidize FGly to produce Ama. Bioinformatic study revealed that 3-CyFEs have evolved from a common ancestor with anaerobic sulfatase maturases (anSMEs), and possess a similar set of catalytic residues with anSMEs. Remarkably, the enzyme does not need leader peptide for activity and is fully active on a truncated peptide containing only 5 amino acids of the core sequence. Our work discloses the first ribosomal path towards Ama formation, providing a possible hint for the rich occurrence of Ama in Nature.

Variants in the ethylmalonyl-CoA decarboxylase (ECHDC1) gene: a novel player in ethylmalonic aciduria?

Ethylmalonic acid (EMA) is a major and potentially cytotoxic metabolite associated with short-chain acyl-CoA dehydrogenase (SCAD) deficiency, a condition whose status as a disease is uncertain. Unexplained high EMA is observed in some individuals with complex neurological symptoms, who carry the SCAD gene (ACADS) variants, c.625G>A and c.511C>T. The variants have a high allele frequency in the general population, but are significantly overrepresented in individuals with elevated EMA. This has led to the idea that these variants need to be associated with variants in other genes to cause hyperexcretion of ethylmalonic acid and possibly a diseased state. Ethylmalonyl-CoA decarboxylase (ECHDC1) has been described and characterized as an EMA metabolite repair enzyme, however, its clinical relevance has never been investigated. In this study, we sequenced the ECHDC1 gene (ECHDC1) in 82 individuals, who were reported with unexplained high EMA levels due to the presence of the common ACADS variants only. Three individuals with ACADS c.625G>A variants were found to be heterozygous for ECHDC1 loss-of-function variants. Knockdown experiments of ECHDC1, in healthy human cells with different ACADS c.625G>A genotypes, showed that ECHDC1 haploinsufficiency and homozygosity for the ACADS c.625G>A variant had a synergistic effect on cellular EMA excretion. This study reports the first cases of ECHDC1 gene defects in humans and suggests that ECHDC1 may be involved in elevated EMA excretion in only a small group of individuals with the common ACADS variants. However, a direct link between ECHDC1/ACADS deficiency, EMA and disease could not be proven.

Malonate utilization by Pseudomonas aeruginosa affects quorum-sensing and virulence and leads to formation of mineralized biofilm-like structures.

Pseudomonas aeruginosa is an opportunistic pathogen that uses malonate among its many carbon sources. We recently reported that, when grown in blood from trauma patients, P. aeruginosa expression of malonate utilization genes was upregulated. In this study, we explored the role of malonate utilization and its contribution to P. aeruginosa virulence. We grew P. aeruginosa strain PA14 in M9 minimal medium containing malonate (MM9) or glycerol (GM9) as a sole carbon source and assessed the effect of the growth on quorum sensing, virulence factors, and antibiotic resistance. Growth of PA14 in MM9, compared to GM9, reduced the production of elastases, rhamnolipids, and pyoverdine; enhanced the production of pyocyanin and catalase; and increased its sensitivity to norfloxacin. Growth in MM9 decreased extracellular levels of N-acylhomoserine lactone autoinducers, an effect likely associated with increased pH of the culture medium; but had little effect on extracellular levels of PQS. At 18 hr of growth in MM9, PA14 formed biofilm-like structures or aggregates that were associated with biomineralization, which was related to increased pH of the culture medium. These results suggest that malonate significantly impacts P. aeruginosa pathogenesis by influencing the quorum sensing systems, the production of virulence factors, biofilm formation, and antibiotic resistance.

Identification of the rhizospheric microbe and metabolites that led by the continuous cropping of ramie (Boehmeria nivea L. Gaud).

Continuous cropping lowers the production and quality of ramie (Boehmeria nivea L. Gaud). This study aimed to reveal the metagenomic and metabolomic changes between the healthy- and obstacle-plant after a long period of continuous cropping. After 10 years of continuous cropping, ramie planted in some portions of the land exhibited weak growth and low yield (Obstacle-group), whereas, ramie planted in the other portion of the land grew healthy (Health-group). We collected rhizosphere soil and root samples from which measurements of soil chemical and plant physiochemical properties were taken. All samples were subjected to non-targeted gas chromatograph-mass spectrometer (GS/MS) metabolome analysis. Further, metagenomics was performed to analyze the functional genes in rhizospheric soil organisms. Based on the findings, ramie in Obstacle-group were characterized by shorter plant height, smaller stem diameter, and lower fiber production than that in Health-group. Besides, the Obstacle-group showed a lower relative abundance of Rhizobiaceae, Lysobacter antibioticus, and Bradyrhizobium japonicum, but a higher relative abundance of Azospirillum lipoferum and A. brasilense compared to the Health-group. Metabolomic analysis results implicated cysteinylglycine (Cys-Gly), uracil, malonate, and glycerol as the key differential metabolites between the Health- and Obstacle-group. Notably, this work revealed that bacteria such as Rhizobia potentially synthesize IAA and are likely to reduce the biotic stress of ramie. L. antibioticus also exerts a positive effect on plants in the fight against biotic stress and is mediated by metabolites including orthophosphate, uracil, and Cys-Gly, which may serve as markers for disease risk. These bacterial effects can play a key role in plant resistance to biotic stress via metabolic and methionine metabolism pathways.

A Korean child diagnosed with malonic aciduria harboring a novel start codon mutation following presentation with dilated cardiomyopathy.

BACKGROUND: Malonic aciduria (MA, OMIM#248360) is an extremely rare inherited metabolic disorder caused by the deficiency of malonyl-CoA decarboxylase. The phenotype exhibited by patients with MA is variable, but may include symptoms, such as developmental delay in early childhood, seizures, vomiting, metabolic acidosis, hypoglycemia, ketosis, and cardiomyopathy. We describe the first case of a Korean child with MA who presented with dilated cardiomyopathy (DCMP) at the age of 3 months. METHODS AND RESULTS: A 3-month-old Korean boy visited our hospital for diagnosis and management of cardiomegaly. Newborn screening for inherited metabolic diseases showed a normal result; therefore, DCMP management was initiated. Biochemical and the MLYCD gene analyses subsequently confirmed diagnosis of MA. Elevated plasma C3DC level and excessive excretion of urinary malonate were observed, and two pathogenic variants, including a novel start codon mutation (c.1A>G), were identified in MLYCD. A low long-chain fat diet with middle-chain triglyceride formula and L-carnitine supplementation was initiated. The patient is now 5 years old and exhibits considerably improved cardiac function. CONCLUSIONS: MA can be diagnosed using newborn screening; however, negative results do not exclude the possibility of disease. Metabolic screening for differential diagnosis of infantile DCMP is recommended to rule out rare, but manageable, metabolic cardiomyopathies.

Structural snapshots of the minimal PKS system responsible for octaketide biosynthesis.

Type II polyketide synthases (PKSs) are multi-enzyme complexes that produce secondary metabolites of medical relevance. Chemical backbones of such polyketides are produced by minimal PKS systems that consist of a malonyl transacylase, an acyl carrier protein and an α/ß heterodimeric ketosynthase. Here, we present X-ray structures of all ternary complexes that constitute the minimal PKS system for anthraquinone biosynthesis in Photorhabdus luminescens. In addition, we characterize this invariable core using molecular simulations, mutagenesis experiments and functional assays. We show that malonylation of the acyl carrier protein is accompanied by major structural rearrangements in the transacylase. Principles of an ongoing chain elongation are derived from the ternary complex with a hexaketide covalently linking the heterodimeric ketosynthase with the acyl carrier protein. Our results for the minimal PKS system provide mechanistic understanding of PKSs and a fundamental basis for engineering PKS pathways for future applications.

Identification of histone malonylation in the human fetal brain and implications for diabetes-induced neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are severe congenital malformations. Diabetes during pregnancy is a risk factor for NTDs, but its mechanism remains elusive. Emerging evidence suggests that protein malonylation is involved in diabetes. Here, we report the correlation between histone lysine malonylation in diabetes-induced NTDs. METHODS: Nano-HPLC/MS/MS was used to screen the histone malonylation profile in human embryonic brain tissue. Then, the histone malonylation level was compared between the brains of normal control mice and mice with diabetes-induced NTDs. Finally, the histone malonylation level was compared under high glucose exposure in an E9 neuroepithelial cell line (NE4C). RESULTS: A total of 30 histone malonylation sites were identified in human embryonic brain tissue, including 18 novel sites. Furthermore, we found an increased histone malonylation level in brain tissues from mice with diabetes-induced NTDs. Finally, both the histone malonylation modified sites and the modified levels were proved to be increased in the NE4C treated with high glucose. CONCLUSION: Our results present a comprehensive map of histone malonylation in the human fetal brain. Furthermore, we provide experimental evidence supporting a relationship between histone malonylation and NTDs caused by high glucose-induced diabetes. These findings offer new insights into the pathological role of histone modifications in human NTDs.

Synthesis and mass spectra of rearrangement bio-signature metabolites of anaerobic alkane degradation via fumarate addition.

Metabolite profiling in anaerobic alkane biodegradation plays an important role in revealing activation mechanisms. Apart from alkylsuccinates, which are considered to be the usual biomarkers via fumarate addition, the downstream metabolites of C-skeleton rearrangement can also be regarded as biomarkers. However, it is difficult to detect intermediate metabolites in both environmental samples and enrichment cultures, resulting in lacking direct evidence to prove the occurrence of fumarate addition pathway. In this work, a synthetic method of rearrangement metabolites was established. Four compounds, namely, propylmalonic acid, 2-(2-methylbutyl)malonic acid, 2-(2-methylpentyl)malonic acid and 2-(2-methyloctyl)malonic acid, were synthesized and determined by four derivatization approaches. Besides, their mass spectra were obtained. Four characteristic ions were observed at m/z 133 + 14n, 160 + 28n, 173 + 28n and [M - (45 + 14n)]+ (n = 0 and 2 for ethyl and n-butyl esters, respectively). For methyl esterification, mass spectral features were m/z 132, 145 and [M - 31]+, while for silylation, fragments were m/z 73, 147, 217, 248, 261 and [M - 15]+. These data provide basis on identification of potential rearrangement metabolites in anaerobic alkane biodegradation via fumarate addition.