Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular ...kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence.
Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning.
In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.
•The glucokinase α8-helix is important for enzyme stability and kinetics.•MODY2 mutations L306R and L309P prevent GKRP-mediated nuclear import of glucokinase.•The MODY2 mutation L304P inactivates the glucokinase nuclear export signal.•The function of the glucokinase nuclear export signal depends on exportin 1.
A total of 1,220 subjects from Equatorial Guinea living in Spain (median age = 41 years; 453 male and 767 female) was examined for antibodies to human immunodeficiency virus (HIV) and Hepatitis B ...(HBV), C (HCV), and D (HDV) viruses. Extracted RNA and DNA from the positive samples were used to quantify viral load. The prevalence of HIV antibodies, HCV RNA, and HBV surface antigen (HBsAg) was 10.8% (N = 132), 11.6% (N = 141), and 7.9% (N = 96), respectively. The most prevalent HIV variant was CRF02_AG (38.5%; N = 40). HCV genotype 4 (60%; N = 36) and HBV genotype A3 (32%; N = 8) were the hepatitis variants most frequently found. Superinfection with HDV was seen in 20.9% (N = 24) of HBsAg carriers. A control group of 276 immigrants from other sub-Saharan countries showed similar rates of HIV and HBsAg, although no HCV cases were found. Immigrants constitute a major source of HIV and hepatitis viruses in Spain; therefore, it is important that control measures are intensified.
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 (S(0.5) = 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.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Immigration from developing regions to Western countries has resulted in an increased rate of non-B subtypes in the HIV population. However, it is unclear whether these HIV variants remain confined ...to foreigners or are already spreading among natives. Since many immigrants come from regions in which hepatitis B virus (HBV) and hepatitis C virus (HCV) are endemic, HIV-hepatitis coinfection might be more frequent in newly diagnosed HIV persons. Herein, we report changes in the prevalence and distribution of HIV-1 subtypes in Madrid, Spain over the past 10 years as well as the rate of chronic HBV and HCV coinfection in 1854 newly diagnosed HIV-1 individuals. Overall 18.2% carried HIV-1 non-B subtypes, although the prevalence increased over time reaching a peak of 19.4% in the last period (2007-2010). The most common non-B variants were CRF02_AG (37%), G (12%), A (9.9%), and C (7.8%). In native Spaniards the rate of non-B subtypes increased from 1.5% in 2000-2002 to 7.2% in 2003-2006 and to 11.4% in 2007-2010 (p = 0.04). Chronic hepatitis B and C were found, respectively, in 4.2% and 8.3% of the study population. While the prevalence of chronic hepatitis B has remained fairly stable over time across distinct populations, the rate of chronic HCV infection has experienced a significant decline, mainly in native Spaniards as a result of a reduction in intravenous drug use. In summary, the prevalence of HIV-1 non-B subtypes is rising in newly diagnosed HIV-1 individuals in Spain, including the native population. In contrast, the rate of HBV coinfection remains unchanged and the rate of HCV coinfection has declined.
Background: Hypoglycaemic drugs that close the KATP channel have been tested in patients with permanent neonatal diabetes due to glucokinase mutations (PNDM-GCK). From the results obtained, it has ...been suggested that this treatment may be beneficial in patients carrying GCK mutations with mild kinetic defects. The aim of this study was to evaluate the kinetic analysis of glucokinase activity as a predictive factor for response to sulphonylureas in PNDM-GCK.
Methods: The clinical characteristics of two siblings with PNDM born to non-consanguineous parents are described. Mutation analysis of KCNJ11, INS and GCK genes was done by sequencing. A comprehensive functional characterisation of GCK mutation was undertaken. Glibenclamide treatment was assayed for 16 weeks in one child. Response to treatment was evaluated by means of fasting glycaemia, C-peptide and HbA1c levels.
Results: Compound heterozygous GCK mutations (p.Ile19Asn and p.Ser441Trp) were identified. Functional analysis of GCK(p.Ile19Asn) indicated that this mutant retained more than 70% of wild-type catalytic activity in vitro, with a slight increase of thermolability. This mutation did not impair the interaction with the glucokinase regulatory protein, and the enzymatic activity of the GCK(p.Ile19Asn) mutant is restored to wild-type levels in the presence of GCK allosteric activator LY2121260. However, glibenclamide treatment of the patient on a reduced dose of insulin did not reduce HbA1c levels, and C-peptide increased only very slightly.
Conclusion: Hypoglycaemic drugs acting on the KATP channel might not be useful in the treatment of PNDM-GCK, even in patients carrying GCK mutations with mild kinetic defects.
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