Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes

The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a “structural movie” of the catalytic steps. High- to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE.Transmethylation, the transfer of a methyl group from one molecule to another, is a fundamental chemical reaction that plays a central role in important biological processes such as gene expression, cell growth, and apoptosis (1). The highly conserved methionine adenosyltransferase (MAT) enzymes synthesize the main source of methyl groups in all living organisms in the form of S-adenosylmethionine (SAMe) (2). The MAT family of enzymes is conserved throughout the kingdoms, emphasizing both their importance and essential role in maintaining appropriate levels of SAMe.Mammals express three MAT genes: MAT I alpha (MAT1A), MAT II alpha (MAT2A), and MAT II beta (MAT2B). MAT1A and MAT2A encode for the catalytic subunits, MATα1 and MATα2, which share an 84% sequence similarity. MAT2B encodes for MATβ, the regulatory subunit that has low sequence similarity (7%) to the catalytic subunits. MATα and MATβ subunits can form several MATαβ complexes (3, 4). There are two major splicing forms of the MAT2B gene that encode for MATβV1 and MATβV2, which share very high sequence similarity and differ by only 20 residues in the N terminus (5). We recently reported that MATβ isoforms interact through their C terminus with MATα2 to form the MATαβ complexes MATαβV1 and MATαβV2 (4). MATα1 can exist in two oligomeric states, dimer and tetramer; however, there was little information on the oligomeric state of MATα2 until recently, when the structure of MATα2βV2 showed that MATα2 can exist and function as a tetramer in the presence of MATβ (4).Several diseases are known to arise from dysregulation of MAT1A, MAT2A, and MAT2B. For example, mutations involving the MAT1A gene have links with hepatic MAT deficiency which leads to hypermethioninemia (6, 7). Expression of MATα2 confers a growth advantage in cells and is important for differentiation and apoptosis (1) in diseases such as human hepatocellular carcinoma (5), colon cancer (8), and leukemia (9).A diverse series of structures are available for MAT enzymes from bacteria, rat, and human (4, 10–12), which, supported by a range of biochemical evidence, have provided significant insight into the enzymatic mechanism. SAMe synthesis follows an SN₂ catalytic mechanism (13, 14) in which the reaction is initiated through a nucleophilic attack by the sulfur atom of methionine on the C5′ atom of ATP, which produces the intermediate tripolyphosphate (PPPi). Hydrolysis of the PPPi into pyrophosphate (PPi) and orthophosphate (Pi) then occurs (Fig. S1).Open in a separate windowFig. S1.Schematic of the enzymatic reaction of SAMe synthesis. Residues of MATα2 that interact with ATP within the active site are shown; dashed lines indicate hydrogen bonds. (A) The interaction of the amide groups of Asp31 and Lys32 with His29 polarizing its imidazol ring and favouring the N-proton release. (B) The sulphur atom of methionine performs a nucleophilic attack on the C5′ atom of ATP. Arrows indicate the movement of electron pairs. (C) Through a nucleophilic substitution reaction the sulphur atom and the C5′ atom form a new bond creating SAMe as the C5′–O5′ of ATP is cleaved.A common feature of MAT enzymes is a gating loop that flanks the active site (in human MATα2 residues 113–131), which has been postulated to act in a dynamic way to allow access to the active site. It has been suggested that when the active site is occupied, the loop is closed like a gate, but when the active site is empty, the loop becomes invisible in the structure, presumably resulting from an open dynamic gate (11). Whether the opening of the loop/gate is required for the entry of substrate and release of products remains unclear. It has been shown that MAT enzymes not only metabolize methionine, but also, depending on species, can process many methionine analogs (15). Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis in rats (16). MAT enzymes are able to produce S-adenosylethionine (SAE) from ethionine (17), which in turn acts as a competitor of methionine. As a result, insufficient levels of SAMe are produced within the cell, leading to disruption of SAMe-dependent processes, such as nicotinamide catabolism (18). SAE can be used to ethylate targets by donating its ethyl group in direct competition with methylation, which results in abnormal alkylation of nucleic acids (19, 20).Here, for the first time to our knowledge, we report several ligand-bound structures of human MATα2 at 1.1 Å (SAMe+ADO+MET+PPNP), 1.85 Å (SAE), and 2.3 Å [p-nitrophenyl phosphate (PPNP)], providing information on the formation of SAMe and SAE in human MATα2 and supporting the common mechanism that exists among MAT enzymes. A detailed view of the position of the triple phosphate group within the active site clearly reveals the orientation of the three phosphoryl moieties, as well as the placement, coordination, and identity of ions within the active site. The resulting insights into the movement of methionine during catalysis elucidate some of the key catalytic events that lead to product formation. We also report the first, to our knowledge, nonarchaeal MAT enzyme structure containing a nonnative product, SAE, that occupies the active site in a similar manner as SAMe, suggesting a similar mechanism for the utilization of ethionine to form the product SAE.     

Initial clinical experience with extracorporeal shock wave therapy in treatment of ischemic heart failure

Previous experimental studies have suggested a beneficial effect of extracorporeal shock wave therapy (ESWT) in patients with ischemic heart failure. Twenty-four patients with ischemic heart failure and left ventricular ejection fraction (LVEF) <40% received ESWT in addition to their stable treatment. ESWT was performed in 9 sessions with 100 shocks per spot in viable segments detected by dobutamine stress echocardiography. Patients were evaluated at baseline and at 3 and 6 months after ESWT. Tc-99m MIBI single photon emission computed tomography was performed on inclusion and at 6 months. ESWT significantly decreased New York Heart Association class from 2.2±0.8 to 1.7±0.7 at 3 months (P<.01) and 6 months after ESWT (1.7±0.7). Six-minute walk test improved from 414±141 to 509±141 and 538±116 (P<.01) at 3 and 6 months, respectively. A steady decrease of Canadian Cardiovascular Society angina class from 2.6±0.7 to 2.1±0.8 and 1.9±0.7 (P<.01) at 3 and 6 months, respectively, was observed. A significant increase in LVEF at rest at 3 and 6 months after ESWT (from 32.2±6.0 to 34.8±9.6 and 37.7±9.5, P=.03, respectively) was noted. Summed rest score (from 23.9±8.1 to 21.4±7.1, P=.03) and stress score improvement (from 28.2±8.4 to 24.6±6.4, P=.04) by single photon emission computed tomography was registered. Significant clinical improvement accompanied by beneficial changes of LVEF and rest/stress perfusion was found after ESWT.     

Effects of testosterone supplementation on markers of the metabolic syndrome and inflammation in hypogonadal men with the metabolic syndrome: the double‐blinded placebo‐controlled Moscow study

Objective Men with the metabolic syndrome (MetS) have low plasma testosterone (T) levels. The aim of this study was to establish whether the normalization of plasma T improves the features of the MetS. Design A randomized, placebo‐controlled, double‐blinded, phase III trial of 184 men suffering from both the MetS and hypogonadism. Patients One hundred and eighty‐four men, aged 35–70, with the MetS and hypogonadism (baseline total T level <12·0 nm or calculated free T level <225 pm .), recruited in the outpatient andrology and urology clinic, Research Center for Endocrinology in Moscow, Russia. Intervention Treatment for 30 weeks with either parenteral T undecanoate (n = 113; TU; 1000 mg IM) or placebo (n = 71), administered at baseline, and after 6 and 18 weeks. One hundred and five (92·9%) men receiving TU and 65 (91·5%) receiving placebo completed the trial. Measurements Body weight, body mass index (BMI), waist circumference (WC), hip circumference, waist‐to‐hip ratio, insulin, leptin, glucose, cholesterol, triglycerides, high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, C‐reactive protein (CRP), interleukin‐1‐beta (IL‐1β), interleukin‐6 (IL‐6), interleukin‐10 (IL‐10) and tumour necrosis factor‐alpha (TNF‐α). Results There were significant decreases in weight, BMI and WC in the TU vs placebo group. Levels of leptin and insulin also decreased, but there were no changes in serum glucose or lipid profile. Of the inflammatory markers, IL‐1β, TNF‐α and CRP decreased, while IL‐6 and IL‐10 did not change significantly. Conclusions Thirty weeks of T administration normalizing plasma T in hypogonadal men with the MetS improved some components of the MetS and a number of inflammatory markers.     

Genetic control of the corticosterone level at rest and under emotional stress in ISIAH rats with inherited stress-induced arterial hypertension

The genetic background of the regulatory systems of the hypothalamic-pituitary-adrenal (HPA) axis in hypertension remains unclear. The inherited stress-induced arterial hypertension (ISIAH) and Wistar Albino Glaxo (WAG) normotensive rats were bred and their F(2) progeny were used in a quantitative trait loci (QTL) analysis to identify genomic regions for plasma basal and stress-induced corticosterone levels, and for absolute and relative adrenal gland weights. The significant loci were found for stress-induced corticosterone on chromosome 9 and for adrenal weight on chromosome 6. The results may help to identify the genes controlling the trait phenotypes in the ISIAH rats characterized by the enhanced responsiveness to stressful stimulation.     

A technical note on endonasal combined microscopic endoscopic with free head navigation technique of removal of pituitary adenomas

Pituitary surgery exemplifies the continuous refinement of surgical techniques. The transsphenoidal approach is the approach of choice to treat most pituitary adenomas. We report here, as a technical note, an operative nuance that represents an encompassment of various technical steps that we utilize in our current surgery, including the corroboration of navigation system on a free head with combined use of endoscope and microscope techniques.     

Basal Insulin Requirements on Continuous Subcutaneous Insulin Infusion During the First 12 Months After Diagnosis of Type 1 Diabetes Mellitus

Introduction

While the endogenous first-phase insulin response has disappeared by the time of diagnosis of type 1 diabetes mellitus (T1DM), anecdotal evidence suggests that these patients can continue to have a second-phase insulin response during the first 12 months after diagnosis. We hypothesized that patients who are started on continuous subcutaneous insulin infusion (CSII) at the time of diagnosis of T1DM would have a lower basal insulin requirement than the 40-60% usually expected.

Methods

We analyzed 38 patients with T1DM, age 9.9 ± 6.4 years, 71% male, who were started on CSII within the first month of diagnosis.

Results

Average basal insulin requirements were 47–49% of total daily dose during the first 12 months after diagnosis and decreased from 0.30 U/kg/day at diagnosis to 0.20 U/kg/day by 12 months. Baseline percentage of basal insulin was significantly correlated with hemoglobin A1c at baseline and at six months. The percentage of basal insulin requirement at 12 months after diagnosis was significantly correlated with baseline body mass index (BMI) and current BMI. No other correlations between percentage of basal insulin requirements and any other factors were seen.

Conclusion

Our data suggest that, even though some endogenous insulin production remains during the first year after diagnosis of T1DM, the distribution of basal versus total daily insulin requirements remains the same as in the general population of people with diabetes. There may be benefits to starting patients on a higher basal rate at time of diagnosis for overall glycemic control during the first six months. Further research is needed to optimize starting insulin doses to maximize their potential in preserving beta-cell function.     

Incidence of nonspecific and radiating low back pain: followup of 24–39‐year‐old adults of the Young Finns Study

Objective

The full panorama of the types and severity of low back pain during the life course is poorly known. We studied the incidence and severity of nonspecific and radiating low back pain based on a recent followup on a representative sample of the Finnish general population.

Methods

As part of the ongoing Young Finns Study, participants ages 24–39 years who were free from low back pain at baseline in 2001 were included (n = 1,224). We estimated the incidence of nonspecific and radiating low back pain in 2007.

Results

The incidence of moderate (8–30 days duration in the past 12 months) nonspecific low back pain was 13.2%, and that of radiating low back pain was 8.6%. The rates did not differ between men and women. The incidence of major (>30 days in the past 12 months) nonspecific low back pain was 7.8%, and that of radiating low back pain was 3.4%. The incidence of major nonspecific low back pain was higher in women than in men (P = 0.02). Moderate as well as major nonspecific low back pain declined with age, whereas major radiating low back pain increased with age.

Conclusion

Our findings indicate that low back pain is a common condition in adults already in their 30s. It becomes more severe around the age of 40 years, showing different development of nonspecific and radiating low back pain. We recommend monitoring low back health in health surveillance, starting early and differentiating between nonspecific and radiating pain.     

REM sleep characteristics of nightmare sufferers before and after REM sleep deprivation

ObjectivesTo examine whether disrupted regulation of REM sleep propensity is implicated in nightmare (NM) pathophysiology.BackgroundHeightened REM propensity induced by REM sleep deprivation is belied by increases in REM %, REM density and the dreamlike quality of dream mentation during post-deprivation recovery sleep. Compromised regulation of REM sleep propensity may be a contributing factor in the pathophysiology of frequent NMs.MethodsA preliminary study of 14 subjects with frequent NMs (?1 NM/week; 27.6 ± 9.9 years) and 11 healthy control subjects (<1 NM/month; 24.3 ± 5.3 years) was undertaken. Subjects completed home sleep/dream logs and underwent three nights of polysomnographic recording with REM sleep deprivation on night 2. Group differences were assessed for a battery of REM sleep and dream measures on nights 1 and 3.ResultsSeveral measures, including #skipped early-night REM periods, REM latency, REM/NREM cycle length, early/late REM density,REM rebound, late-night REM% and dream vividness, suggested that REM sleep propensity was abnormally low for the frequent NM group throughout the 3-day study.ConclusionsFindings raise the possibility that REM anomalies recorded from NM sufferers sleeping in the laboratory environment reflect a disruption of one or more endogenous regulators of REM sleep propensity.