Removal Of Methionine Research Articles

Cloning of the Thermus thermophilus methionine aminopeptidase gene and confirmation of the enzyme functional activity

Background. Methionine aminopeptidases (MAPs) are a class of enzymes that catalyze the removal of the N-terminal initiator methionine from a polypeptide chain. Bacterial MAPs are considered as targets for the development of broad-spectrum antibacterial drugs, and using MAPs in biotechnology necessitates the search for new MAPs and the study of their functioning and inhibition mechanisms.The aim of the study. To identify methionine aminopeptidase in the Thermus thermophilus genome (Tt-MAP) and to confirm its functional activity.Materials and methods. To identify Tt-MAP, we analyzed the Thermus thermophilus genome in the GeneBank database. Modern genetic engineering techniques (polymerase chain reaction, restriction, transformation, heterologous expression) were used to clone the putative open reading frame (ORF) encoding Tt-MAP in the pHUE vector. Various chromatography techniques (affinity, ion exchange, and size-exclusion) were used to obtain a purified enzyme preparation. The fluorogenic substrate L-methionine 7-amino-4-methylcoumarin (Met-AMC) was used to confirm the specific functional aminopeptidase activity of the enzyme.Results. An ORF encoding MAP was identified in the Thermus thermophilus bacterium genome. Oligonucleotide primers were designed based on the nucleotide sequence. The ORF was cloned in the vector, and the recombinant enzyme was produced in E. coli cells. A method for purifying the enzyme to a homogeneous state was developed using a series of sequential chromatographies, allowing up to 30 mg to be obtained from 1 liter of culture. Using the fluorogenic substrate Met-AMC, the specific functional activity of the enzyme was demonstrated (the enzyme cleaves methionine from the substrate).Conclusion. We have identified the Thermus thermophilus MAP and tested its functional activity. It has been shown that the ORF product TTHA1670 encodes a methionine-specific aminopeptidase, i. e. methionine aminopeptidase. The enzyme can be used in various fields of biotechnology and scientific research.

Discovery of anti-tumor agent targeted MetAP-2 using a special turn-on fluorescent probe

Methionine aminopeptidase 2 (MetAP-2) catalyzes the removal of methionine from the amino-terminus of proteins or peptides and is a known target for anti-tumor therapy. In this study, we developed an enzyme-activated fluorescent probe, DDAO-BMT, specifically for the real-time detection of endogenous MetAP-2. This probe enables the determination of extracellular MetAP-2 activity and visual monitoring of intracellular MetAP-2. Additionally, Sappan Lignum was identified as an active herbal medicine against MetAP-2, with brazilin identified as its key active constituent. Brazilin also exhibited a significant anti-tumor effect, including the inhibition on proliferation, migration, and invasion of tumor cells, and the preliminary mechanism of its activity against cancer has been elucidated. The use of this probe facilitates the monitoring of endogenous MetAP-2 in complex biological system, aiding in the discovery of novel active compounds for cancer treatment.

Komrower Memorial Lecture 2023. Molecular basis of phenotype expression in homocystinuria: Where are we 30 years later?

This review summarises progress in the research of homocystinuria (HCU) in the past three decades. HCU due to cystathionine β-synthase (CBS) was discovered in 1962, and Prof. Jan Peter Kraus summarised developments in the field in the first-ever Komrower lecture in 1993. In the past three decades, significant advancements have been achieved in the biology of CBS, including gene organisation, tissue expression, 3D structures, and regulatory mechanisms. Renewed interest in CBS arose in the late 1990s when this enzyme was implicated in biogenesis of H2S. Advancements in genetic and biochemical techniques enabled the identification of several hundreds of pathogenic CBS variants and the misfolding of missense mutations as a common mechanism. Several cellular, invertebrate and murine HCU models allowed us to gain insights into functional and metabolic pathophysiology of the disease. Establishing the E-HOD consortium and patient networks, HCU Network Australia and HCU Network America, offered new possibilities for acquiring clinical data in registries and data on patients' quality of life. A recent analysis of data from the E-HOD registry showed that the clinical variability of HCU is broad, extending from severe childhood disease to milder (late) adulthood forms, which typically respond to pyridoxine. Pyridoxine responsiveness appears to be the key factor determining the clinical course of HCU. Increased awareness about HCU played a role in developing novel therapies, such as gene therapy, correction of misfolding by chaperones, removal of methionine from the gut and enzyme therapies that decrease homocysteine or methionine in the circulation.

Identification of a Cryptic Pocket in Methionine Aminopeptidase-II Using Adaptive Bandit Molecular Dynamics Simulations and Markov State Models.

Methionine aminopeptidase-II (MetAP-II) is a metalloprotease, primarily responsible for the cotranslational removal of the N-terminal initiator methionine from the nascent polypeptide chain during protein synthesis. MetAP-II has been implicated in angiogenesis and endothelial cell proliferation and is therefore considered a validated target for cancer therapeutics. However, there is no effective drug available against MetAP-II. In this study, we employ Adaptive Bandit molecular dynamics simulations to investigate the structural dynamics of the apo and ligand-bound MetAP-II. Our results focus on the dynamic behavior of the disordered loop that is not resolved in most of the crystal structures. Further analysis of the conformational flexibility of the disordered loop reveals a hidden cryptic pocket that is predicted to be potentially druggable. The network analysis indicates that the disordered loop region has a direct signaling route to the active site. These findings highlight a new way to target MetAP-II by designing inhibitors for the allosteric site within this disordered loop region.

Obtaining Overstable Methionine Aminopeptidase for the Removal of Methionine From Recombinant Proteins

Cleavage of the N-terminal initiating methionine (Met1) is a critical coand post-translational modification affecting 50–70% of cellular proteins. During the production of recombinant proteins in the heterologous system of E. coli expression, Met1 cleavage often fails to occur, which leads to heterogeneity of the preparations obtained, changes in their activity and stability. This problem can be solved by treating recombinant proteins in vitro with a specific enzyme, methionine aminopeptidase (MAP). Currently available MAPs exhibit limited specificities and reaction conditions. We cloned a MAP from a hyperthermophilic bacterium, developed a method for enzyme purification, and studied a number of physicochemical properties. The new MAP enzyme is resistant to elevated temperatures. The MAP maintains a stable native state in a pH range from 3 to 11 units. The novel MAP enzyme can be used to remove N-terminal Met1 from recombinant proteins in vitro over a wide pH range and at elevated temperatures.

Responses in splanchnic and mammary amino acid metabolism to short-term graded removal of methionine in lactating goats.

Four multi-catheterized lactating goats were used in a 4×4 Latin square experiment to investigate the responses of amino acid metabolism in portal-drained viscera (PDV), liver, and mammary glands to short-term varying supplies of methionine (Met). During the last 45h in each experimental period, goats were fasted for 12h and then abomasally infused with an amino acid (AA) mixture plus glucose for 33h. Treatments consisted of graded removal of Met from an infused AA mixture to achieve Met content in the infusate of 100% (complete), 60%, 30%, or 0% that in casein. Graded Met removal decreased the production of milk, milk protein, lactose, and fat linearly whilst also decreasing arterial Met concentration linearly (P<0.05). Meanwhile, net PDV uptake and liver removal of Met decreased linearly (P<0.05) due to decreased Met affinity of PDV and liver (P<0.05). Net mammary uptake of Met (P>0.1) was maintained as Met supply declined. This was achieved through increased mammary affinity (P<0.05) and increased mammary blood flow (P<0.05) totally offsetting the negative effect of decreased circulating Met concentration. Graded removal of Met from the infusate linearly decreased mammary uptake-to-milk output ratios of Met (P<0.05) and tended to decrease essential amino acid (EAA) linearly (0.05<P<0.1). Treatments also linearly decreased circulating concentration of prolactin and linearly increased insulin concentration (P<0.05). In conclusion, results of the present study indicated there were several mechanisms used to mitigate a Met deficiency, including reduced catabolism of Met in PDV, liver, and peripheral tissue (including mammary glands) and a linear increase in mammary blood flow. The observed decreases in milk protein production as Met supply decreased appear to be a result of regulatory events which may have been driven by decreased circulating prolactin, rather than as a result of decreased mammary Met uptake.

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Protein complexes are the functional machines in the cell and are heterogeneous due to protein sequence variations and post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering the microheterogeneity of intact protein complexes. The method exhibited superior separation resolution for protein complexes than conventional native capillary zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations and MS characterization of seven different forms of a streptavidin homotetramer with variations of N-terminal methionine removal, acetylation, and formylation and four forms of the carbonic anhydrase-zinc complex arising from variations of PTMs (succinimide, deamidation, etc.). In addition, ncIEF-MS resolved different states of an interchain cysteine-linked antibody-drug conjugate (ADC1) as a new class of anticancer therapeutic agents that bears a distribution of varied drug-to-antibody ratio (DAR) species. More importantly, ncIEF-MS enabled precise measurements of isoelectric points (pIs) of protein complexes, which reflect the surface electrostatic properties of protein complexes. We studied how protein sequence variations/PTMs modulate the pIs of protein complexes and how drug loading affects the pIs of antibodies. We discovered that keeping the N-terminal methionine residue of one subunit of the streptavidin homotetramer decreased its pI by 0.1, adding one acetyl group onto the streptavidin homotetramer reduced its pI by nearly 0.4, incorporating one formyl group onto the streptavidin homotetramer reduced its pI by around 0.3, and loading two more drug molecules on one ADC1 molecule increased its pI by 0.1. The data render the ncIEF-MS method a valuable tool for delineating protein complexes.

To New Beginnings: Riboproteogenomics Discovery of N-Terminal Proteoforms in Arabidopsis Thaliana.

Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.

CBMS-10 Methionine metabolism closely related with self-renew, pluripotency and cell death in GICs through modification of cholesterol biosynthesis and ribosomal RNA

Glioma initiating cells (GICs) are the source of glioma cells that have the ability to self-renew and pluripotency, which are treatment-resistant, starting point for relapse and eventual death despite multimodality therapy. Since high accumulation is observed in 11cMet-PET at the time of recurrence, it is important to understand the mechanism of tumor cell activation caused by the reorganization of methionine metabolism. We cultured cells in methionine-deprived culture medium and performed a comprehensive analysis, and found that methionine depletion markedly decreased proliferation and increasing cell death of GICs. Decreased SAM, which is synthesized intracellularly catalyzed by methionine adenosyltransferase (MAT) using methionine, triggered the following: (i) global DNA demethylation, (ii) hyper-methylation of signaling pathways regulating pluripotentcy of stem cells, (iii) decreased expression of the core-genes and pluripotent marker of stem cells including FOXM1, SOX2, SOX4, PROM1 and OLIG2, (iv) decreased cholesterol synthesis and increased excretion mainly through decreased SREBF2 (v) down-regulation of the large subunit of ribosomal protein configured 28S and ACA43, snoRNA guiding the pseudouridylation of 28S ribosomal RNA, which has crucial role for translation. In addition, inhibition of cholesterol synthesis with statin resulted in a phenotype similar to that of methionine removal and a decrease in stem cell markers and snoRNA ACA43. Moreover, suppression of FOXM1 decreased stem cell markers such as SOX4 and PROM1. The gene expression profile for cholesterol production was obtained from the Ivy Glioblastoma Atlas Project (IVYGAP) database and compared between tumour cells with relatively low methionine levels in area of pseudopalisading arrangement around necrosis and tumour cells in the infiltrating region, showing that cells cells in the infiltrating region have a higher capacity to produce cholesterol. Taken together, methionine metabolism closely related with self-renew, pluripotency and cell death in GICs through modification of cholesterol biosynthesis: especially SREBF2-FOXM1 and ACA43 axis with modification of ribosomal RNA.

Selective inhibition of Helicobacter pylori methionine aminopeptidase by azaindole hydroxamic acid derivatives: Design, synthesis, in vitro biochemical and structural studies

Methionine aminopeptidases (MetAPs) are an important class of enzymes that work co-translationally for the removal of initiator methionine. Chemical inhibition or gene knockdown is lethal to the microbes suggesting that they can be used as antibiotic targets. However, sequence and structural similarity between the microbial and host MetAPs has been a challenge in the identification of selective inhibitors. In this study, we have analyzed several thousands of MetAP sequences and established a pattern of variation in the S1 pocket of the enzyme. Based on this knowledge, we have designed a library of 17 azaindole based hydroxamic acid derivatives which selectively inhibited the MetAP from H. pylori compared to the human counterpart. Structural studies provided the molecular basis for the selectivity.

Removal of N-terminal methionine of human interferon α-2b by co‐producing with Pyrococcus furiosus methionine aminopeptidase in Escherichia coli

In our previous studies, expression of human interferon α-2b gene in Escherichia coli gave three types of polypeptide chains in nearly equimolar ratio. They include molecules with N-terminal methionine, molecules without N-terminal methionine, and molecules with acetylated N-terminal. This heterogeneity is not required in therapeutic applications. In order to improve the removal rate of N-terminus methionine, co-expression of human interferon α-2b and methionine aminopeptidase genes was performed in E. coli. The co-expression resulted in production of human interferon α-2b with dominance of molecules without N-terminal methionine and very few of them were acetylated at the N-terminus. Our findings contribute significantly to efforts for the production of homogenous therapeutic proteins which represent the exact recombinant replica of their native proteins.

Cardiac Troponin T: The Impact of Posttranslational Modifications on Analytical Immunoreactivity in Blood up to the Excretion in Urine.

Cardiac troponin T (cTnT) is a sensitive and specific biomarker for detecting cardiac muscle injury. Its concentration in blood can be significantly elevated outside the normal reference range under several pathophysiological conditions. The classical analytical method in routine clinical analysis to detect cTnT in serum or plasma is a single commercial immunoassay, which is designed to quantify the intact cTnT molecule. The targeted epitopes are located in the central region of the cTnT molecule. However, in blood cTnT exists in different biomolecular complexes and proteoforms: bound (to cardiac troponin subunits or to immunoglobulins) or unbound (as intact protein or as proteolytic proteoforms). While proteolysis is a principal posttranslational modification (PTM), other confirmed PTMs of the proteoforms include N-terminal initiator methionine removal, N-acetylation, O-phosphorylation, O-(N-acetyl)-glucosaminylation, N(ɛ)-(carboxymethyl)lysine modification and citrullination. The immunoassay probably detects several of those cTnT biomolecular complexes and proteoforms, as long as they have the centrally targeted epitopes in common. While analytical cTnT immunoreactivity has been studied predominantly in blood, it can also be detected in urine, although it is unclear in which proteoform cTnT immunoreactivity is present in urine. This review presents an overview of the current knowledge on the pathophysiological lifecycle of cTnT. It provides insight into the impact of PTMs, not only on the analytical immunoreactivity, but also on the excretion of cTnT in urine as one of the waste routes in that lifecycle. Accordingly, and after isolating the proteoforms from urine of patients suffering from proteinuria and acute myocardial infarction, the structures of some possible cTnT proteoforms are reconstructed using mass spectrometry and presented.

KRAS(G12C)\u2013AMG\xa0510 interaction dynamics revealed by all-atom molecular dynamics simulations

The first KRAS(G12C) targeting inhibitor in clinical development, AMG 510, has shown promising antitumor activity in clinical trials. On the molecular level, however, the interaction dynamics of this covalently bound drug–protein complex has been undetermined. Here, we disclose the interaction dynamics of the KRAS(G12C)–AMG 510 complex by long timescale all-atom molecular dynamics (MD) simulations (total of 75 μs). Moreover, we investigated the influence of the recently reported post-translational modification (PTM) of KRAS’ N-terminus, removal of initiator methionine (iMet1) with acetylation of Thr2, to this complex. Our results demonstrate that AMG 510 does not entrap KRAS into a single conformation, as one would expect based on the crystal structure, but rather into an ensemble of conformations. AMG 510 binding is extremely stable regardless of highly dynamic interface of KRAS’ switches. Overall, KRAS(G12C)–AMG 510 complex partially mimic the native dynamics of GDP bound KRAS; however, AMG 510 stabilizes the α3-helix region. N-terminally modified KRAS displays similar interaction dynamics with AMG 510 as when Met1 is present, but this PTM appears to stabilize β2–β3-loop. These results provide novel conformational insights on the molecular level to KRAS(G12C)–AMG 510 interactions and dynamics, providing new perspectives to RAS related drug discovery.

In Vitro and In Vivo Characterization of Potent Antileishmanial Methionine Aminopeptidase 1 Inhibitors.

Leishmania major is the causative agent of cutaneous leishmaniasis (CL). No human vaccine is available for CL, and current drug regimens present several drawbacks, such as emerging resistance, severe toxicity, medium effectiveness, and/or high cost. Thus, the need for better treatment options against CL is a priority. In the present study, we validate the enzyme methionine aminopeptidase 1 of L. major (MetAP1Lm), a metalloprotease that catalyzes the removal of N-terminal methionine from peptides and proteins, as a chemotherapeutic target against CL infection. The in vitro antileishmanial activities of eight novel MetAP1 inhibitors (OJT001 to OJT008) were investigated. Three compounds, OJT006, OJT007, and OJT008, demonstrated potent antiproliferative effects in macrophages infected with L. major amastigotes and promastigotes at submicromolar concentrations, with no cytotoxicity against host cells. Importantly, the leishmanicidal effect in transgenic L. major promastigotes overexpressing MetAP1Lm was diminished by almost 10-fold in comparison to the effect in wild-type promastigotes. Furthermore, the in vivo activities of OJT006, OJT007, and OJT008 were investigated in L. major-infected BALB/c mice. In comparison to the footpad parasite load in the control group, OJT008 decreased the footpad parasite load significantly, by 86%, and exhibited no toxicity in treated mice. We propose MetAP1 inhibitor OJT008 as a potential chemotherapeutic candidate against CL infection caused by L. major infection.

Comprehensive Characterization of the Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase by Top-Down Mass Spectrometry.

Reversible phosphorylation plays critical roles in cell growth, division, and signal transduction. Kinases which catalyze the transfer of γ-phosphate groups of nucleotide triphosphates to their substrates are central to the regulation of protein phosphorylation and are therefore important therapeutic targets. Top-down mass spectrometry (MS) presents unique opportunities to study protein kinases owing to its capabilities in comprehensive characterization of proteoforms that arise from alternative splicing, sequence variations, and post-translational modifications. Here, for the first time, we developed a top-down MS method to characterize the catalytic subunit (C-subunit) of an important kinase, cAMP-dependent protein kinase (PKA). The recombinant PKA C-subunit was expressed in Escherichia coli and successfully purified via his-tag affinity purification. By intact mass analysis with high resolution and high accuracy, four different proteoforms of the affinity-purified PKA C-subunit were detected, and the most abundant proteoform was found containing seven phosphorylations with the removal of N-terminal methionine. Subsequently, the seven phosphorylation sites of the most abundant PKA C-subunit proteoform were characterized simultaneously using tandem MS methods. Four sites were unambiguously identified as Ser10, Ser11, Ser18, and Ser30, and the remaining phosphorylation sites were localized to Ser2/Ser3, Ser358/Thr368, and Thr[215-224]Tyr in the PKA C-subunit sequence with a 20mer 6xHis-tag added at the N-terminus. Interestingly, four of these seven phosphorylation sites were located at the 6xHis-tag. Furthermore, we have performed dephosphorylation reaction by Lambda protein phosphatase and showed that all phosphorylations of the recombinant PKA C-subunit phosphoproteoforms were removed by this phosphatase.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics.

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.

Short-term lactation and mammary metabolism responses in lactating goats to graded removal of methionine from an intravenously infused complete amino acid mixture

To investigate the possible pathways of Met deficiency to depress milk protein synthesis, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h followed by a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period, and mammary biopsy was performed in the last hour. Treatments were graded removal of Met from the infused AA mixture to achieve Met content in the infusate of 100 (complete), 60, 30, or 0% of that in casein. Graded Met removal decreased yield of milk, milk protein, and lactose linearly and tended to decrease yield of milk fat linearly. Milk protein yield decreased to 82, 78, and 69% that of complete mixture infusion, respectively, when the 60, 30, and 0% Met infusate was infused. Circulating Met decreased linearly with graded Met removal. Arterial and venous Met decreased to 36 and 23% that of complete mixture infusion, respectively, when all Met was removed out of the mixture. Concomitant with the decreased circulating concentration was a similar increase in mammary Met affinity as reflected by the linearly increased mammary Met clearance rate. The increased affinity plus the linearly increased mammary blood flow totally offset the negative effect of decreased circulating Met concentration on mammary Met uptake. The overall result was similar mammary Met uptakes across treatments ranging from 285.9 to 339.5 μmol/h. Mammary uptakes of the other AA measured were generally not affected by treatments except for a linearly decreased Thr uptake and a trend of linearly increased Glu uptake. Consistent with the behavior of an AA mainly catabolized in the liver and mainly used for protein synthesis in peripheral tissues, mammary uptake to milk output ratios of Met measured in the present study ranged from 1.25 to 1.49 and was not affected by treatments. For the other AA measured, the ratio of Thr was linearly decreased and that of Glu was linearly increased by graded Met removal. Graded Met removal linearly elevated circulating urea N and glucose concentrations, indicating enhanced whole-body catabolism of AA and hepatic gluconeogenesis. Treatments had no significant effects on circulating insulin, growth hormone, and the other hormones and metabolites measured. Phosphorylation status of eIF4E binding protein 1 tended to decrease linearly and that of p70S6k was linearly decreased by graded Met removal, indicating depressed signal in the intracellular mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. In conclusion, results of the present study indicated that the mTORC1 pathway and whole-body AA catabolism rather than mammary uptake appeared the drivers for changes in milk protein synthesis in response to varying Met supply.

In vivo methylation of OLA1 revealed by activity-based target profiling of NTMT1.

N-Terminal methyltransferase 1 (NTMT1) catalyzes the N-terminal methylation of proteins with a specific N-terminal motif after methionine removal. Aberrant N-terminal methylation has been implicated in several cancers and developmental diseases. Together with motif sequence and signal peptide analyses, activity-based substrate profiling of NTMT1 utilizing (E)-hex-2-en-5-ynyl-S-adenosyl-l-methionine (Hey-SAM) revealed 72 potential targets, which include several previously confirmed ones and many unknowns. Target validation using normal and NTMT1 knock-out (KO) HEK293FT cells generated by CRISPR-Cas9 demonstrated that Obg-like ATPase 1 (OLA1), a protein involved in many critical cellular functions, is methylated in vivo by NTMT1. Additionally, Hey-SAM synthesis achieved ≥98% yield for SAH conversion.

Role of dietary amino acid balance in diet restriction-mediated lifespan extension, renoprotection, and muscle weakness in aged mice.

SummaryExtending healthy lifespan is an emerging issue in an aging society. This study was designed to identify a dietary method of extending lifespan, promoting renoprotection, and preventing muscle weakness in aged mice, with a focus on the importance of the balance between dietary essential (EAAs) and nonessential amino acids (NEAAs) on the dietary restriction (DR)‐induced antiaging effect. Groups of aged mice were fed ad libitum, a simple DR, or a DR with recovering NEAAs or EAAs. Simple DR significantly extended lifespan and ameliorated age‐related kidney injury; however, the beneficial effects of DR were canceled by recovering dietary EAA but not NEAA. Simple DR prevented the age‐dependent decrease in slow‐twitch muscle fiber function but reduced absolute fast‐twitch muscle fiber function. DR‐induced fast‐twitch muscle fiber dysfunction was improved by recovering either dietary NEAAs or EAAs. In the ad libitum‐fed and the DR plus EAA groups, the renal content of methionine, an EAA, was significantly higher, accompanied by lower renal production of hydrogen sulfide (H2S), an endogenous antioxidant. Finally, removal of methionine from the dietary EAA supplement diminished the adverse effects of dietary EAA on lifespan and kidney injury in the diet‐restricted aged mice, which were accompanied by a recovery in H2S production capacity and lower oxidative stress. These data imply that a dietary approach could combat kidney aging and prolong lifespan, while preventing muscle weakness, and suggest that renal methionine metabolism and the trans‐sulfuration pathway could be therapeutic targets for preventing kidney aging and subsequently promoting healthy aging.

Corneal Endothelial Cells Have an Absolute Requirement for Cysteine for Survival.

The aim of this study was to evaluate which amino acid(s) among the 20 standard protein amino acids is indispensable for the survival of cultured human corneal endothelial cells (HCECs). HCECs were cultured in amino acid screening media that were missing 1 specific amino acid, and cell growth was evaluated. After this first selection, we conducted a further evaluation of cell growth in response to the addition of 4 amino acids (cysteine, methionine, valine, and arginine) to amino acid-free culture media. We then evaluated the antioxidant effect of cysteine compared with other antioxidants in terms of apoptosis of HCECs, rabbit corneal endothelial cell (CECs), monkey CECs, and ex vivo human donor corneas. Culture in an amino acid-free Dulbecco Modified Eagle Medium (DMEM) decreased the cell numbers to 11.0% when compared with culture in normal DMEM. Removal of cysteine, methionine, valine, or arginine from DMEM significantly suppressed cell numbers (27.7%, 61.4%, 75.5%, and 60.6%, respectively) (P < 0.01), whereas removal of other amino acids did not significantly decrease cell numbers. A lack of cysteine induced apoptosis, but addition of antioxidants reversed this. Removal of cysteine induced in vitro apoptosis in HCECs, rabbit CECs, monkey CECs, and ex vivo human donor corneas, whereas the presence of cysteine almost completely suppressed this apoptosis. Cysteine seems to be an indispensable amino acid for HCEC growth and survival. Its necessity might reflect a high requirement for antioxidants to protect HCECs from oxidative stress, as HCECs have high aerobic metabolic activity.