Knockout of Tmlhe in mice is not associated with autism spectrum disorder phenotypes or motor dysfunction despite low carnitine levels.

Deletion of exon 2 of the trimethyllysine hydroxylase epsilon (TMLHE) gene was identified in probands with autism spectrum disorder (ASD). TMLHE encodes the first enzyme in carnitine biosynthesis, N6-trimethyllysine dioxygenase (TMLD). Researchers have suggested that carnitine depletion could be important for the development of ASD and cognitive, locomotor and social dysfunctions, but previous findings have been inconclusive regarding the specific role of endogenous carnitine. We developed a mouse knockout model with constitutive TMLD enzyme inactivation that exhibited a significant decrease in the carnitine by more than 90% compared to wild-type (WT) mice. However, we did not observe any significant social, cognitive, or repetitive-behavior changes associated with ASD in the knockout mice; muscle strength and coordination were also not affected. In addition, the life expectancy of knockout mice was similar to that of WT mice. In conclusion, knockout of Tmlh in mice does not induce an ASD phenotype or motor dysfunction despite extremely low carnitine and gamma-butyrobetaine concentrations. Moreover, inactivation of TMLD does not induce a phenotype similar to previously described primary carnitine deficiency; indeed, our results showed that low levels of carnitine sustained adequate energy production, muscle function and social behavior in mice.

γ-enolase (ENO2) is methylated at the Nτ position of His-190 among enolase isozymes.

Protein methylation is mainly observed in lysine, arginine and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18 and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between the brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.

Neither Trimethylamine-N-Oxide nor Trimethyllysine Is Associated with Atherosclerosis: A Cross-Sectional Study in Older Japanese Adults.

Recent evidence suggests that trimethylamine-N-oxide (TMAO), a metabolite of L-carnitine and choline, is linked to atherosclerosis and cardiovascular diseases. As TMAO content is very high in fish, we raised the following question: why do Japanese people, who consume lots of fish, show a low risk of atherosclerosis? To address this question, we investigated the effects of TMAO and other L-carnitine-related metabolites on carotid intima-media thickness (IMT). Participants were recruited from a small island and a mountainous region. Plasma L-carnitine, γ-butyrobetaine (γBB), TMAO, trimethyllysine (TML), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) levels were measured using liquid or gas chromatography-mass spectrometry. Plasma L-carnitine concentration was higher in men than in women. TMAO and TML were significantly higher in the residents of the island than in the mountainous people. In multiple linear regression analyses in all participants, TML showed a significant inverse association with max-IMT and plaque score (PS), whereas TMAO did not show any associations. In women, L-carnitine was positively associated with max-IMT and PS. TMAO was correlated with both EPA and DHA levels, implying that fish is a major dietary source of TMAO in Japanese people. Our study found that plasma TMAO was not an apparent risk factor for atherosclerosis in elderly Japanese people, whereas a low level of TML might be a potential risk. L-carnitine may be a marker for atherosclerosis in women.

Trimethyllysine, a trimethylamine N-oxide precursor, predicts the presence, severity, and prognosis of heart failure.

Background and aims: Intestinal flora metabolites are associated with cardiovascular (CV) diseases including heart failure (HF). The carnitine precursor trimethyllysine (TML), which participates in the generation of the atherogenic-related metabolite trimethylamine N-oxide (TMAO), was found to be related to poor prognosis in patients with CV diseases. The aim of the present study was to examine the relationship between TML and stable chronic HF. Methods and results: In total, 956 subjects including 471 stable chronic HF and 485 non-HF patients were enrolled in the present cohort study and subjects with stable HF were followed up for 2.0 ± 1.1 years. Serum levels of TML and TMAO were measured by liquid chromatography mass spectrometry in tandem. TML levels were significantly elevated in patients with HF compared with non-HF patients and were positively correlated with N-terminal pro-brain natriuretic peptide (NTproBNP) levels (r = 0.448, P < 0.001). TML was associated with the presence of HF after adjusting for age, sex, complications, traditional clinical factors, and TMAO (tertile 3 (T3), adjusted odds ratio (OR) 1.93, 95% confidence interval (CI) 1.19-3.13, and P = 0.007). In patients with HF, increased TML levels were associated with a composite endpoint of CV death and HF hospitalization during follow-up (T3, adjusted hazard ratio (HR) 1.93, 95% CI 1.27-2.93, and P = 0.002). Increased TML levels indicated a higher risk of CV death, re-hospitalization, and all-cause mortality. Conclusion: Serum TML levels were associated with the presence and severity of HF in all subjects. High levels of TML can indicate complications and poor prognosis in HF patients.

Association of Systemic Trimethyllysine With Heart Failure With Preserved Ejection Fraction and Cardiovascular Events.

CONTEXT: Carnitine has been associated with cardiac energy metabolism and heart failure, but the association between its precursors-trimethyllysine (TML) and γ-butyrobetaine (GBB)-and heart failure with preserved ejection fraction (HFpEF) remains unclear. OBJECTIVE: To evaluate the relationship between TML-related metabolites and HFpEF in an Asian population. METHODS: The cross-sectional component of this study examined the association between plasma TML-related metabolites and HFpEF, while a prospective cohort design was applied to examine the association with incident cardiovascular events in HFpEF. Included in the study were 1000 individuals who did not have heart failure (non-HF) and 1413 patients with HFpEF. Liquid chromatography mass spectrometry was used to assess plasma carnitine, GBB, TML and trimethylamine-N-oxide (TMAO) concentrations. RESULTS: Plasma GBB and TML were both elevated in patients with HFpEF. After adjusting for traditional risk factors and renal function, TML, but not GBB, was significantly associated with HFpEF. The odds ratio (OR) for the fourth vs first quartile of TML was 1.57 (95% CI 1.09-2.27; P-trend < .01). The OR for each SD increment of log-TML was 1.26 (95% CI 1.08-1.47). Plasma TMAO (P-interaction = 0.024) and estimated glomerular filtration rate (P-interaction = 0.024) modified the TML-HFpEF association. The addition of TML improved the diagnostic value under the multivariable model. In the prospective study of patients with HFpEF, higher plasma TML was associated with increased risk of cardiovascular events. CONCLUSION: Plasma TML concentrations are positively associated with HFpEF, and higher plasma TML indicates increased risk of cardiovascular events.

Association Between Plasma Trimethyllysine and Prognosis of Patients With Ischemic Stroke.

Background Trimethyllysine, a trimethylamine N-oxide precursor, has been identified as an independent cardiovascular risk factor in acute coronary syndrome. However, limited data are available to examine the role of trimethyllysine in the population with stroke. We aimed to examine the relationship between plasma trimethyllysine levels and stroke outcomes in patients presenting with ischemic stroke or transient ischemic attack. Methods and Results Data of 10 027 patients with ischemic stroke/transient ischemic attack from the CNSR-III (Third China National Stroke Registry) and 1-year follow-up data for stroke outcomes were analyzed. Plasma levels of trimethyllysine were measured with mass spectrometry. The association between trimethyllysine and stroke outcomes was analyzed using Cox regression models. Mediation analysis was performed to examine the mediation effects of risk factors on the associations of trimethyllysine and stroke outcomes. Elevated trimethyllysine levels were associated with increased risk of cardiovascular death (quartile 4 versus quartile 1: adjusted hazard ratio [HR], 1.72; 95% CI, 1.03-2.86) and all-cause mortality (quartile 4 versus quartile 1: HR, 1.97; 95% CI, 1.40-2.78) in multivariate Cox regression model. However, no associations were found between trimethyllysine and nonfatal stroke recurrence or nonfatal myocardial infarction. Trimethyllysine was associated with cardiovascular death independent of trimethylamine N-oxide. Both estimated glomerular filtration rate and hs-CRP (high-sensitivity C-reactive protein) had significant mediation effects on the association of trimethyllysine with cardiovascular death, with a mediation effect of 37.8% and 13.4%, respectively. Conclusions Elevated trimethyllysine level is associated with cardiovascular death among patients with ischemic stroke/transient ischemic attack. Mediation analyses propose that trimethyllysine contributes to cardiovascular death through inflammation and renal function, suggesting a possible pathomechanistic link.

Low cardiac content of long-chain acylcarnitines in TMLHE knockout mice prevents ischaemia-reperfusion-induced mitochondrial and cardiac damage.

Increased tissue content of long-chain acylcarnitines may induce mitochondrial and cardiac damage by stimulating ROS production. N6-trimethyllysine dioxygenase (TMLD) is the first enzyme in the carnitine/acylcarnitine biosynthesis pathway. Inactivation of the TMLHE gene (TMLHE KO) in mice is expected to limit long-chain acylcarnitine synthesis and thus induce a cardio- and mitochondria-protective phenotype. TMLHE gene deletion in male mice lowered acylcarnitine concentrations in blood and cardiac tissues by up to 85% and decreased fatty acid oxidation by 30% but did not affect muscle and heart function in mice. Metabolome profile analysis revealed increased levels of polyunsaturated fatty acids (PUFAs) and a global shift in fatty acid content from saturated to unsaturated lipids. In the risk area of ischemic hearts in TMLHE KO mouse, the OXPHOS-dependent respiration rate and OXPHOS coupling efficiency were fully preserved. Additionally, the decreased long-chain acylcarnitine synthesis rate in TMLHE KO mice prevented ischaemia-reperfusion-induced ROS production in cardiac mitochondria. This was associated with a 39% smaller infarct size in the TMLHE KO mice. The arrest of the acylcarnitine biosynthesis pathway in TMLHE KO mice prevents ischaemia-reperfusion-induced damage in cardiac mitochondria and decreases infarct size. These results confirm that the decreased accumulation of ROS-increasing fatty acid metabolism intermediates prevents mitochondrial and cardiac damage during ischaemia-reperfusion.

The Capacity of APOB-Depleted Plasma in Inducing ATP-Binding Cassette A1/G1-Mediated Macrophage Cholesterol Efflux-But Not Gut Microbial-Derived Metabolites-Is Independently Associated with Mortality in Patients with ST-Segment Elevation Myocardial Infarction.

Impaired HDL-mediated macrophage cholesterol efflux and higher circulating concentrations of trimethylamine N-oxide (TMAO) levels are independent risk factors for cardiovascular mortality. The TMAO precursors, γ-butyrobetaine (γBB) and Trimethyllysine (TML), have also been recently associated with cardiovascular death, but their interactions with HDL-mediated cholesterol efflux remain unclear. We aimed to determine the associations between APOB depleted plasma-mediated macrophage cholesterol efflux and plasma TMAO, γBB, and TML concentrations and explore their association with two-year follow-up mortality in patients with acute ST-elevation myocardial infarction (STEMI) and unstable angina (UA). Baseline and ATP-binding cassette transporter ABCA1 and ABCG1 (ABCA1/G1)-mediated macrophage cholesterol efflux to APOB-depleted plasma was decreased in patients with STEMI, and the latter was further impaired in those who died during follow-up. Moreover, the circulating concentrations of TMAO, γBB, and TML were higher in the deceased STEMI patients when compared with the STEMI survivors or UA patients. However, after statistical adjustment, only ABCA1/G1-mediated macrophage cholesterol efflux remained significantly associated with mortality. Furthermore, neither the TMAO, γBB, nor TML levels altered the HDL-mediated macrophage cholesterol efflux in vitro. We conclude that impaired ABCA1/G1-mediated macrophage cholesterol efflux is independently associated with mortality at follow-up in STEMI patients.

L-Carnitine Production Through Biosensor-Guided Construction of the Neurospora crassa Biosynthesis Pathway in Escherichia coli.

L-Carnitine is a bioactive compound derived from L-lysine and S-adenosyl-L-methionine, which is closely associated with the transport of long-chain fatty acids in the intermediary metabolism of eukaryotes and sought after in the pharmaceutical, food, and feed industries. The L-carnitine biosynthesis pathway has not been observed in prokaryotes, and the use of eukaryotic microorganisms as natural L-carnitine producers lacks economic viability due to complex cultivation and low titers. While biotransformation processes based on petrochemical achiral precursors have been described for bacterial hosts, fermentative de novo synthesis has not been established although it holds the potential for a sustainable and economical one-pot process using renewable feedstocks. This study describes the metabolic engineering of Escherichia coli for L-carnitine production. L-carnitine biosynthesis enzymes from the fungus Neurospora crassa that were functionally active in E. coli were identified and applied individually or in cascades to assemble and optimize a four-step L-carnitine biosynthesis pathway in this host. Pathway performance was monitored by a transcription factor-based L-carnitine biosensor. The engineered E. coli strain produced L-carnitine from supplemented L-N ε-trimethyllysine in a whole cell biotransformation, resulting in 15.9 µM carnitine found in the supernatant. Notably, this strain also produced 1.7 µM L-carnitine de novo from glycerol and ammonium as carbon and nitrogen sources through endogenous N ε-trimethyllysine. This work provides a proof of concept for the de novo L-carnitine production in E. coli, which does not depend on petrochemical synthesis of achiral precursors, but makes use of renewable feedstocks instead. To the best of our knowledge, this is the first description of L-carnitine de novo synthesis using an engineered bacterium.

Trimethyllysine, vascular risk factors and outcome in acute ischemic stroke (MARK-STROKE).

Trimethyllysine (TML) is involved in the generation of the pro-atherogenic metabolite trimethylamine-N-oxide (TMAO) by gut microbiota. In clinical studies, elevated TML levels predicted major adverse cardiovascular events (MACE) in patients with acute or stable coronary artery disease (CAD). In contrast to cardiovascular patients, the role of TML in patients with acute cerebral ischemia is unknown. Here, we evaluated circulating TML levels in 374 stroke patients from the prospective biomarkers in stroke (MARK-STROKE) study. Compared with 167 matched healthy controls, acute ischemic stroke patients had lower median TML plasma concentrations, i.e. 0.71 vs. 0.47 µmol/L (p < 0.001) and this difference persisted after adjusting for age and sex. TML plasma concentrations were associated with age, serum creatinine, glucose, cholesterol and lysine. Patients with prevalent arterial hypertension, atrial fibrillation or a history of myocardial infarction had increased TML levels, but this observation was not independent of age, sex and GFR. In 274 patients, follow-up data were available. During a median follow-up of 284 [25th-75th percentile: 198, 431] days, TML was not associated with incident MACE (stroke, myocardial infarction, death). In summary, our data suggests a different role of TML in acute ischemic stroke compared with CAD patients.

Trimethyllysine: From Carnitine Biosynthesis to Epigenetics.

Trimethyllysine is an important post-translationally modified amino acid with functions in the carnitine biosynthesis and regulation of key epigenetic processes. Protein lysine methyltransferases and demethylases dynamically control protein lysine methylation, with each state of methylation changing the biophysical properties of lysine and the subsequent effect on protein function, in particular histone proteins and their central role in epigenetics. Epigenetic reader domain proteins can distinguish between different lysine methylation states and initiate downstream cellular processes upon recognition. Dysregulation of protein methylation is linked to various diseases, including cancer, inflammation, and genetic disorders. In this review, we cover biomolecular studies on the role of trimethyllysine in carnitine biosynthesis, different enzymatic reactions involved in the synthesis and removal of trimethyllysine, trimethyllysine recognition by reader proteins, and the role of trimethyllysine on the nucleosome assembly.

Carnitine Inborn Errors of Metabolism.

Carnitine plays essential roles in intermediary metabolism. In non-vegetarians, most of carnitine sources (~75%) are obtained from diet whereas endogenous synthesis accounts for around 25%. Renal carnitine reabsorption along with dietary intake and endogenous production maintain carnitine homeostasis. The precursors for carnitine biosynthesis are lysine and methionine. The biosynthetic pathway involves four enzymes: 6-N-trimethyllysine dioxygenase (TMLD), 3-hydroxy-6-N-trimethyllysine aldolase (HTMLA), 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABADH), and γ-butyrobetaine dioxygenase (BBD). OCTN2 (organic cation/carnitine transporter novel type 2) transports carnitine into the cells. One of the major functions of carnitine is shuttling long-chain fatty acids across the mitochondrial membrane from the cytosol into the mitochondrial matrix for ß-oxidation. This transport is achieved by mitochondrial carnitine-acylcarnitine cycle, which consists of three enzymes: carnitine palmitoyltransferase I (CPT I), carnitine-acylcarnitine translocase (CACT), and carnitine palmitoyltransferase II (CPT II). Carnitine inborn errors of metabolism could result from defects in carnitine biosynthesis, carnitine transport, or mitochondrial carnitine-acylcarnitine cycle. The presentation of these disorders is variable but common findings include hypoketotic hypoglycemia, cardio(myopathy), and liver disease. In this review, the metabolism and homeostasis of carnitine are discussed. Then we present details of different inborn errors of carnitine metabolism, including clinical presentation, diagnosis, and treatment options. At the end, we discuss some of the causes of secondary carnitine deficiency.

Trimethyllysine, a trimethylamine N-oxide precursor, provides near- and long-term prognostic value in patients presenting with acute coronary syndromes.

AIMS: Trimethyllysine (TML) serves as a nutrient precursor of the gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) and is associated with incident cardiovascular (CV) events in stable subjects. We examined the relationship between plasma TML levels and incident CV events in patients presenting with acute coronary syndromes (ACS). METHODS AND RESULTS: Plasma levels of TML were quantified in two independent cohorts using mass spectrometry, and its relationship with CV events was investigated. In a Cleveland Cohort (N = 530), comprised of patients presenting to the emergency department with chest pain and suspected ACS, TML was associated with major adverse cardiac events (MACE, myocardial infarction, stroke, need for revascularization, or all-cause mortality) over both 30 days [3rd tertile (T3), adjusted odds ratio (OR) 1.77, 95% confidence interval (CI) 1.04-3.01; P < 0.05] and 6 months (T3, adjusted OR 1.95, 95% CI 1.15-3.32; P < 0.05) of follow-up independent of traditional CV risk factors and indices of renal function. Elevated TML levels were also associated with incident long-term (7-year) all-cause mortality [T3, adjusted hazard ratio (HR) 2.52, 95% CI 1.50-4.24; P < 0.001], and MACE even amongst patients persistently negative for cardiac Troponin T at presentation (e.g. 30-day MACE, T3, adjusted OR 4.49, 95% CI 2.06-9.79; P < 0.001). Trimethyllysine in combination with TMAO showed additive significance for near- and long-term CV events, including patients with 'negative' high-sensitivity Troponin T levels. In a multicentre Swiss Cohort (N = 1683) comprised of ACS patients, similar associations between TML and incident 1-year adverse cardiac risks were observed (e.g. mortality, adjusted T3 HR 2.74, 95% CI 1.28-5.85; P < 0.05; and MACE, adjusted T3 HR 1.55, 95% CI 1.04-2.31; P < 0.05). CONCLUSION: Plasma TML levels, alone and together with TMAO, are associated with both near- and long-term CV events in patients with chest pain and ACS.

Carnitine Precursors and Short-Chain Acylcarnitines in Water Buffalo Milk.

Ruminants' milk contains δ-valerobetaine originating from rumen through the transformation of dietary Nε-trimethyllysine. Among ruminant's milk, the occurrence of δ-valerobetaine, along with carnitine precursors and metabolites, has not been investigated in buffalo milk, the second most worldwide consumed milk, well-known for its nutritional value. HPLC-ESI-MS/MS analyses of bulk milk revealed that the Italian Mediterranean buffalo milk contains δ-valerobetaine at levels higher than those in bovine milk. Importantly, we detected also γ-butyrobetaine, the l-carnitine precursor, never described so far in any milk. Of interest, buffalo milk shows higher levels of acetylcarnitine, propionylcarnitine, butyrylcarnitine, isobutyrylcarnitine, and 3-methylbutyrylcarnitine (isovalerylcarnitine) than cow milk. Moreover, buffalo milk shows isobutyrylcarnitine and butyrylcarnitine at a 1-to-1 molar ratio, while in cow's milk this ratio is 5 to 1. Results indicate a peculiar short-chain acylcarnitine profile characterizing buffalo milk, widening the current knowledge about its composition and nutritional value.

Ruminant meat and milk contain δ-valerobetaine, another precursor of trimethylamine N-oxide (TMAO) like γ-butyrobetaine.

Quaternary ammonium compounds containing N-trimethylamino moiety, such as choline derivatives and carnitine, abundant in meat and dairy products, are metabolic precursors of trimethylamine (TMA). A similar fate is reported for Nε-trimethyllysine and γ-butyrobetaine. With the aim at investigating the metabolic profile of such metabolites in most employed animal dietary sources, HPLC-ESI-MS/MS analyses on ruminant and non-ruminant milk and meat were performed. Results demonstrate, for the first time, the presence of δ-valerobetaine, occurring at levels higher than γ-butyrobetaine in all ruminant samples compared to non-ruminants. Demonstration of δ-valerobetaine metabolic origin, surprisingly, showed that it originates from rumen through the transformation of dietary Nε-trimethyllysine. These results highlight our previous findings showing the ubiquity of free Nε-trimethyllysine in vegetable kingdom. Furthermore, δ-valerobetaine, similarly to γ-butyrobetaine, can be degraded by host gut microbiota producing TMA, precursor of the proatherogenic trimethylamine N-oxide (TMAO), unveiling its possible role in the biosynthetic route of TMAO.

Cardiolipin dynamics and binding to conserved residues in the mitochondrial ADP/ATP carrier.

Cardiolipin in eukaryotes is found in the mitochondrial inner membrane, where it interacts with membrane proteins and, although not essential, is necessary for the optimal activity of a number of proteins. One of them is the mitochondrial ADP/ATP carrier, which imports ADP into the mitochondrion and exports ATP. In the crystal structures, cardiolipin is bound to three equivalent sites of the ADP/ATP carrier, but its role is unresolved. Conservation of residues at these cardiolipin binding sites across other members of the mitochondrial carrier superfamily indicates cardiolipin binding is likely to be important for the function of all mitochondrial carriers. Multiscale simulations were performed in a cardiolipin-containing membrane to investigate the dynamics of cardiolipin around the yeast and bovine ADP/ATP carriers in a lipid bilayer and the properties of the cardiolipin-binding sites. In coarse-grain simulations, cardiolipin molecules bound to the carriers for longer periods of time than phosphatidylcholine and phosphatidylethanolamine lipids-with timescales in the tens of microseconds. Three long-lived cardiolipin binding sites overlapped with those in the crystal structures of the carriers. Other shorter-lived cardiolipin interaction sites were identified in both membrane leaflets. However, the timescales of the interactions were of the same order as phosphatidylcholine and phosphatidylethanolamine, suggesting that these sites are not specific for cardiolipin binding. The calculation of lipid binding times and the overlap of the cardiolipin binding sites between the structures and simulations demonstrate the potential of multiscale simulations to investigate the dynamics and behavior of lipids interacting with membrane proteins.

The betaine profile of cereal flours unveils new and uncommon betaines.

We report the LC-ESI-MS/MS determination of betaines in commercial flours of cereals and pseudocereals most utilized in human nutrition. Results showed that glycine betaine, trigonelline, proline betaine, Nε-trimethyllysine were metabolites common to all examined flours, whereas an uncommon betaine, valine betaine, and glutamine betaine were present only in flours of barley, rye, oat, durum wheat, winter wheat, Triticum dicoccum and Triticum monococcum. Valine betaine and glutamine betaine, the latter never reported before in plants and animals, are not evenly distributed in the Poaceae family, but their presence or absence in flours depends on the subfamily to which the plant belongs. Interestingly, we also report for the first time the occurrence of pipecolic acid betaine (homostachydrine) and its precursor 1,2-N-methylpipecolic acid in rye flour. These two metabolites were not detected in any other cereal or pseudocereal flour, suggesting their potential role as markers of rye flour occurrence in cereal-based foods.

Aza-amino acid scanning of chromobox homolog 7 (CBX7) ligands.

An aza-amino acid scan of peptide inhibitors of the chromobox homolog 7 (CBX7) was performed to study the conformational requirements for affinity to the methyllysine reader protein. Twelve azapeptide analogues were prepared using three different approaches employing respectively N-(Fmoc)aza-amino acid chlorides and submonomer azapeptide synthesis to install systematically aza-residues at the first four residues of the peptide, as well as to provide aza-lysine residues possessing saturated and unsaturated side chains. The aza-peptide ligands were evaluated in a chromobox homolog 7 binding assay, providing useful insight into structural requirements for affinity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases.

The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme.

Supramolecular Ligands for Histone Tails by Employing a Multivalent Display of Trisulfonated Calix[4]arenes.

Post-translational modification of histone tails plays critical roles in gene regulation. Thus, molecules recognizing histone tails and controlling their epigenetic modification are desirable as biochemical tools to elucidate regulatory mechanisms. There are, however, only a few synthetic ligands that bind to histone tails with substantial affinity. We report CA2 and CA3, which exhibited sub-micromolar affinity to histone tails (especially tails with a trimethylated lysine). Multivalent display of trisulfonated calix[4]arene was important for strong binding. CA2 was applicable not only to synthetic tail peptides but also to endogenous histone proteins, and was successfully used to pull-down endogenous histones from nuclear extract. These findings indicate the utility of these supramolecular ligands as biochemical tools for studying chromatin regulator protein and as a targeting motif in ligand-directed catalysis to control epigenetic modifications.