Functional Characterization of MODY2 Mutations Highlights the Importance of the Fine-Tuning of Glucokinase and Its Role in Glucose Sensing

Glucokinase (GK) acts as a glucose sensor in the pancreatic beta-cell and regulates insulin secretion. Heterozygous mutations in the human GK-encoding GCK gene that reduce the activity index increase the glucose-stimulated insulin secretion threshold and cause familial, mild fasting hyperglycaemia, also known as Maturity Onset Diabetes of the Young type 2 (MODY2). Here we describe the biochemical characterization of five missense GK mutations: p.Ile130Thr, p.Asp205His, p.Gly223Ser, p.His416Arg and p.Ala449Thr. The enzymatic analysis of the corresponding bacterially expressed GST-GK mutant proteins show that all of them impair the kinetic characteristics of the enzyme. In keeping with their position within the protein, mutations p.Ile130Thr, p.Asp205His, p.Gly223Ser, and p.His416Arg strongly decrease the activity index of GK, affecting to one or more kinetic parameters. In contrast, the p.Ala449Thr mutation, which is located in the allosteric activator site, does not affect significantly the activity index of GK, but dramatically modifies the main kinetic parameters responsible for the function of this enzyme as a glucose sensor. The reduced Kcat of the mutant (3.21±0.28 s−1 vs 47.86±2.78 s−1) is balanced by an increased glucose affinity (S0.5 = 1.33±0.08 mM vs 7.86±0.09 mM) and loss of cooperativity for this substrate. We further studied the mechanism by which this mutation impaired GK kinetics by measuring the differential effects of several competitive inhibitors and one allosteric activator on the mutant protein. Our results suggest that this mutation alters the equilibrium between the conformational states of glucokinase and highlights the importance of the fine-tuning of GK and its role in glucose sensing

The impact of HIV and antiretroviral therapy on TB risk in children: a systematic review and meta-analysis.

BACKGROUND: Children (<15 years) are vulnerable to TB disease following infection, but no systematic review or meta-analysis has quantified the effects of HIV-related immunosuppression or antiretroviral therapy (ART) on their TB incidence. OBJECTIVES: Determine the impact of HIV infection and ART on risk of incident TB disease in children. METHODS: We searched MEDLINE and Embase for studies measuring HIV prevalence in paediatric TB cases ('TB cohorts') and paediatric HIV cohorts reporting TB incidence ('HIV cohorts'). Study quality was assessed using the Newcastle-Ottawa tool. TB cohorts with controls were meta-analysed to determine the incidence rate ratio (IRR) for TB given HIV. HIV cohort data were meta-analysed to estimate the trend in log-IRR versus CD4%, relative incidence by immunological stage and ART-associated protection from TB. RESULTS: 42 TB cohorts and 22 HIV cohorts were included. In the eight TB cohorts with controls, the IRR for TB was 7.9 (95% CI 4.5 to 13.7). HIV-infected children exhibited a reduction in IRR of 0.94 (95% credible interval: 0.83-1.07) per percentage point increase in CD4%. TB incidence was 5.0 (95% CI 4.0 to 6.0) times higher in children with severe compared with non-significant immunosuppression. TB incidence was lower in HIV-infected children on ART (HR: 0.30; 95% CI 0.21 to 0.39). Following initiation of ART, TB incidence declined rapidly over 12 months towards a HR of 0.10 (95% CI 0.04 to 0.25). CONCLUSIONS: HIV is a potent risk factor for paediatric TB, and ART is strongly protective. In HIV-infected children, early diagnosis and ART initiation reduces TB risk. TRIAL REGISTRATION NUMBER: CRD42014014276

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Effect of temperature on the stability of the GST-GK(p.Ala449Thr) protein.

<p>Stock enzyme solutions were diluted to 250 µg/ml in buffer containing 30% glycerol, 50 mM glucose, 10 mM glutathione, 5 mM dithiothreitol, 200 mM KCl and 50 mM Tris/HCl, pH 8.0. A) The enzyme solutions were incubated for 30 min at different temperatures ranging from 30 to 55°C and then assayed at 30°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>. B) The enzyme solutions were incubated for different periods of time from 5 to 60 min at 50°C. Means and SEM of three independent enzyme preparations are shown.</p

Clinical characteristics of probands and <i>GCK</i> mutations.

<p>BMI: body mass index; FPG: fasting plasma glucose; OGTT: plasma glucose at 120 min after a standard oral glucose tolerance test (1.75 g per kg, máx. 75 g). Nucleotide numbering uses +1 as the A of the ATG initiation codon, based on the GenBank sequence # NM_000162. F, father; M, mother; S, sister; pGF, paternal grandfather; mGF, maternal grandfather; mGM, maternal grandmother. NA, not analysed. Underlined are those affected family members where mutation was checked by genotyping. Novel mutations are shown in bold.</p

Inhibition of GK activity by mannoheptulose (MH), N-acetylglucosamine (NAG) and palmitoyl-CoA.

<p>The GST-GK enzyme was assayed at 5 mM glucose with the indicated concentration of inhibitors as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>. Means and SEM of three independent enzyme preparations are shown.</p

Effect of GK synthetic activator LY2121260 on wild type and mutant GST-GK fusion proteins.

<p>GK activity was measured in standard conditions, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>, in the absence and presence of 10 µM GK activator. Since LY2121260 was dissolved in a buffer containing DMSO, all assays contained a final concentration of 0.8% DMSO. Data represent means ± SEM of at least four separate experiments from two independent enzyme purifications. Statistical significance has been estimated comparing values in the presence of LY2121260 versus their corresponding values in the presence of buffer. (**) <i>p</i><0.005; (*) <i>p</i> = 0.023.</p

Effect of mutation p.Ala449Thr on the GK interaction with GKRP.

<p>A) Inhibition of glucokinase activity by human GKRP. Enzyme activity was measured at 5 mM glucose as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>, in the presence of 10 µM S6P (left panel) or 0.2 mM F1P (right panel). Results are means ± SEM for three independent enzyme purifications assayed in triplicate. B) Two-hybrid interaction of GBD-GKRP with GAD-GK or GAD-GK(p.Ala449Thr) mutant. Yeast strain Y187 was used, and fusion proteins were expressed from pGBKT7 and pACTII derivatives. Values are means ± SEM from ß-galactosidase activity of six independent transformants. In control experiments, GBD-GKRP did not interact with GAD and GAD-GK did not interact with GBD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#pone.0030518-Galan1" target="_blank">[22]</a>.</p

Kinetic constants of human recombinant wild type and MODY2 mutant beta-cell GST-GK fusion proteins.

<p>Data represent means ± SEM of <i>n</i> separate experiments from at least two independent enzyme purifications. The Hill coefficient (nH) and the relative activity index (I_a) are unit less. (*) <i>p</i><0.0007; (**) <i>p</i> = 0.012.</p

Localization of mutations in the structural model for beta-cell glucokinase.

<p>A) Localization of mutated residues in the closed conformation of GK. A to E) An enlargement of the region of interest is shown in each panel. F) Representation of mutation p.Ala449Thr in the super-open conformation of GK. Wild type residues are in black, whereas the mutated residues are magenta. Surrounding disturbing structures are in blue. Glucose is represented in green whereas activator compound A is in yellow. The closed (1V4S) and the super-open (1V4T) conformations of GK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#pone.0030518-Kamata1" target="_blank">[18]</a> are represented using the Pymol Molecular Graphics System (Schrödinger).</p