scholarly journals Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme

1995 ◽  
Vol 309 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Y Liang ◽  
P Kesavan ◽  
L Q Wang ◽  
K Niswender ◽  
Y Tanizawa ◽  
...  
Keyword(s):  
Atp Binding ◽  
Maturity Onset Diabetes ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Induced Fit ◽  
Catalytic Activities ◽  
Onset Diabetes ◽  
Kinetic Effects ◽  
The Stability ◽  
Substrate Affinities

Mutations in the human glucokinase (GK) gene are thought to cause maturity-onset diabetes of youth (MODY) by leading to the production of enzymes with reduced catalytic activities and increased glucose Km values. However, in some cases the diabetic phenotype is more severe than might be predicted from these apparent kinetic effects alone. To determine whether these mutations might also effect other characteristics of the enzyme, nine MODY-associated mutants were expressed as fusion proteins with Schistosoma japonicum glutathione S-transferase (GST) and compared with three wild-type human GK isoforms that were also expressed in the same manner. Three GST-GK isoforms (liver 1, liver 2 and islet) were kinetically indistinguishable from each other and from purified rat liver GK. Noteworthy is a glucose-induced fit effect for the interaction of trinitrophenyl (TNP)-ATP with GST-GK, whereby glucose significantly increased the affinity of TNP-ATP binding to GST-GK without changing the stoichiometry of binding. The nine MODY-associated mutations studied either showed diminished catalytic activity, substrate affinities, allosteric regulation, or stability of the fusion enzyme. We conclude that: (1) Gly261 and Lys414 are important for ATP binding; (2) Val203 may be essential for a glucose-induced fit effect; and (3) the stability of fusion protein may be significantly reduced when Glu300 is replaced by Lys. These results suggest that, in addition to effects on the Km and Vmax. of GK, a decrease in the ATP-binding affinity or stability of the mutated enzyme may also contribute to a reduction of GK activity in individuals with GK-MODY. In the B-cell this would have the effect of blunting glucose-stimulated insulin release, thereby contributing to the diabetic phenotype.

scholarly journals Difference in Stability of the N-domain Underlies Distinct Intracellular Properties of the E1064A and H1069Q Mutants of Copper-transporting ATPase ATP7B

2011 ◽  
Vol 286 (18) ◽  
pp. 16355-16362 ◽  
Author(s):  
Oleg Y. Dmitriev ◽  
Ashima Bhattacharjee ◽  
Sergiy Nokhrin ◽  
Eva-Maria E. Uhlemann ◽  
Svetlana Lutsenko
Keyword(s):  
Phenotypic Variability ◽  
Copper Metabolism ◽  
Complete Loss ◽  
Atp Binding ◽  
Wild Type ◽  
Helical Hairpin ◽  
The Difference ◽  
The Stability ◽  
Golgi Network

Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the α1,α2-helical hairpin (α-HH) that houses Glu1064 and His1069 is altered. The α-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same α-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the α-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.

scholarly journals Structural instability of mutant β-cell glucokinase: implications for the molecular pathogenesis of maturity-onset diabetes of the young (type-2)

1997 ◽  
Vol 322 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Prabakaran KESAVAN ◽  
Liqun WANG ◽  
Elizabeth DAVIS ◽  
Antonio CUESTA ◽  
Ian SWEET ◽  
...  
Keyword(s):  
Structural Instability ◽  
Pancreatic Β Cell ◽  
Maturity Onset Diabetes ◽  
Wild Type ◽  
Β Cell ◽  
Catalytic Function ◽  
Onset Diabetes ◽  
Impaired Insulin ◽  
Thermal Stability Of

The catalytic function and thermal stability of wild-type and mutant recombinant human pancreatic β-cell glucokinase was investigated. The mutants E70K and E300K, which are thought to be the cause of impaired insulin production by the pancreatic β-cell and decreased glucose uptake by the liver of patients with maturity-onset diabetes of the young, were found to be functionally indistinguishable from the wild-type, i.e. their kcat,S0.5, inflection point and hwere normal. However, these two mutants showed markedly reduced stability under a variety of test conditions. Glucokinase instability, not low enzyme catalytic activity, may be the cause of diabetes mellitus with E70K and E300K mutants.

scholarly journals Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability

2005 ◽  
Vol 393 (1) ◽  
pp. 389-396 ◽  
Author(s):  
María Galán ◽  
Olivier Vincent ◽  
Isabel Roncero ◽  
Sharona Azriel ◽  
Pedro Boix-Pallares ◽  
...  
Keyword(s):  
Protein Stability ◽  
Glucose Sensor ◽  
Critical Role ◽  
Regulatory Protein ◽  
Missense Mutations ◽  
Maturity Onset Diabetes ◽  
Glutathione S Transferase ◽  
Enzymatic Assays ◽  
Onset Diabetes ◽  
Healthy Control

Glucokinase acts as the pancreatic glucose sensor and plays a critical role in the regulation of insulin secretion by the β-cell. Heterozygous mutations in the glucokinase-encoding GCK gene, which result in a reduction of the enzymatic activity, cause the monogenic form of diabetes, MODY2 (maturity-onset diabetes of the young 2). We have identified and functionally characterized missense mutations in the GCK gene in diabetic families that result in protein mutations Leu165→Phe, Glu265→Lys and Thr206→Met. The first two are novel GCK mutations that co-segregate with the diabetes phenotype in their respective families and are not found in more than 50 healthy control individuals. In order to measure the biochemical effects of these missense mutations on glucokinase activity, we bacterially expressed and affinity-purified islet human glucokinase proteins carrying the respective mutations and fused to GST (glutathione S-transferase). Enzymatic assays on the recombinant proteins revealed that mutations Thr206→Met and Leu165→Phe strongly affect the kinetic parameters of glucokinase, in agreement with the localization of both residues close to the active site of the enzyme. In contrast, mutation Glu265→Lys, which has a weaker effect on the kinetics of glucokinase, strongly affects the protein stability, suggesting a possible structural defect of this mutant protein. Finally, none of the mutations tested appears to affect the interaction of gluco-kinase with the glucokinase regulatory protein in the yeast two-hybrid system.

Mechanisms of mutual functional interactions between HNF-4α and HNF-1α revealed by mutations that cause maturity onset diabetes of the young

2006 ◽  
Vol 290 (3) ◽  
pp. G466-G475 ◽  
Author(s):  
Christopher W. Rowley ◽  
Lora J. Staloch ◽  
Joyce K. Divine ◽  
Sean P. McCaul ◽  
Theodore C. Simon
Keyword(s):  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Regulatory Sequences ◽  
Maturity Onset Diabetes ◽  
Wild Type ◽  
Fatty Acid Binding ◽  
Functional Interactions ◽  
Onset Diabetes ◽  
Promoter Occupancy

Hepatic nuclear factor (HNF)-4α and HNF-1α are key endodermal transcriptional regulators that physically and functionally interact. HNF-4α and HNF-1α cooperatively activate genes with binding sites for both factors, whereas suppressive interactions occur at regulatory sequences with a binding site for only one factor. The liver fatty acid binding protein gene ( Fabp1) has binding sites for both factors, and chromatin precipitation assays were utilized to demonstrate that HNF-4α increased HNF-1α Fabp1 promoter occupancy during cooperative transcriptional activation. The HNF4 P2 promoter contains a HNF-1 but not HNF-4 binding site, and HNF-4α suppressed HNF-1α HNF4 P2 activation and decreased promoter HNF-1α occupancy. The apolipoprotein C III ( APOC3) promoter contains a HNF-4 but not HNF-1 binding site, and HNF-1α suppressed HNF-4α APOC3 activation and decreased HNF-4α promoter occupancy. Maturity onset diabetes of the young (MODY) as well as defects in hepatic lipid metabolism result from mutations in either HNF-4α or HNF-1α. We found that MODY missense mutant R127W HNF-4α retained wild-type individual Fabp1 activation and bound to HNF-1α better than wild-type HNF-4α, yet did not cooperate with HNF-1α or increase HNF-1α Fabp1 promoter occupancy. The R127W mutant was also defective in both suppressing HNF-1α activation of HNF4 P2 and decreasing HNF-1α promoter occupancy. The HNF-1α R131Q MODY mutant also retained wild-type Fabp1 activation and bound to HNF-4α as well as the wild type but was defective in both suppressing HNF-4α APOC3 activation and decreasing HNF-4α promoter occupancy. These results suggest HNF-1α-HNF-4α functional interactions are accomplished by regulating factor promoter occupancy and that defective factor-factor interactions may contribute to the MODY phenotype.

scholarly journals Cell-biological assessment of human glucokinase mutants causing maturity-onset diabetes of the young type 2 (MODY-2) or glucokinase-linked hyperinsulinaemia (GK-HI)

1999 ◽  
Vol 342 (2) ◽  
pp. 345-352 ◽  
Author(s):  
Charlotte V. BURKE ◽  
Carol W. BUETTGER ◽  
Elizabeth A. DAVIS ◽  
Steven J. MCCLANE ◽  
Franz M. MATSCHINSKY ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Human Islet ◽  
Biological Assessment ◽  
Hill Coefficient ◽  
Published Data ◽  
Mrna Levels ◽  
Maturity Onset Diabetes ◽  
Wild Type ◽  
Onset Diabetes

Mutations in the glucokinase (GK) gene cause type-2 maturity-onset diabetes of the young type 2 (MODY-2) and GK-linked hyperinsulinaemia (GK-HI). Recombinant adenoviruses expressing the human wild-type islet GK or one of four mutant forms of GK, (the MODY-2 mutants E70K, E300K and V203A and the GK-HI mutant V455M) were transduced into glucose-responsive insulin-secreting β-HC9 cells and tested functionally in order to initiate the first analysis in vivoof recombinant wild-type and mutant human islet GK. Kinetic analysis of wild-type human GK showed that the glucose S0.5 and Hill coefficient were similar to previously published data in vitro (S0.5 is the glucose level at the half-maximal rate). E70K had half the glucose affinity of wild-type, but similar enzyme activity. V203A demonstrated decreased catalytic activity and an 8-fold increase in glucose S0.5 when compared with wild-type human islet GK. E300K had a glucose S0.5 similar to wild-type but a 10-fold reduction in enzyme activity. E300K mRNA levels were comparable with wild-type GK mRNA levels, but Western-blot analyses demonstrated markedly reduced levels of immunologically detectable protein, consistent with an instability mutation. V455M was just as active as wild-type GK, but with a markedly reduced S0.5. The effects of the different GK mutants on glucose-stimulated insulin release support the kinetic and expression data. These experiments show the utility of a combined genetic, biochemical and cell-biological approach to the quantification of functional and structural changes of human GK that result from MODY-2 and GK-HI mutations.

Diabetes mellitus bei Kindern und Jugendlichen

2007 ◽  
Vol 7 (04) ◽  
pp. 203-208
Author(s):  
Wieland Kiess ◽  
Thomas Kapellen ◽  
Angela Galler
Keyword(s):  
Diabetes Mellitus ◽  
Diabetes Mellitus Typ 2 ◽  
Maturity Onset Diabetes ◽  
Genetische Diagnostik ◽  
Diabetes Mellitus Typ 1 ◽  
Genetische Faktoren ◽  
Onset Diabetes ◽  
Inherited Diabetes

ZusammenfassungGene spielen bei der Pathogenese des Diabetes mellitus eine wichtige Rolle. Die häufigste Form bei Kindern und Jugendlichen ist der Diabetes mellitus Typ 1. Bei vorhandener genetischer Prädisposition kann durch verschiedene Umweltfaktoren eine Autoimmunreaktion ausgelöst werden, welche durch Zerstörung der Betazellen zum Insulinmangel und somit zum Diabetes mellitus Typ 1 führt. Beim Diabetes mellitus Typ 2, welcher bei der zunehmenden Adipositas im Kindes- und Jugendalter in den letzten Jahren in Deutschland häufiger zu beobachten ist, spielen genetische Faktoren eine entscheidende Rolle. Der Diabetes mellitus Typ 2 wird polygen vererbt. Bisher liegen jedoch nur unzureichende Daten vor, um eine genetische Diagnostik in der Praxis sinnvoll erscheinen zu lassen. Bei einer Reihe von weiteren Diabetestypen ist deren genetische Ursache in den letzten Jahrzehnten geklärt worden. Eine genetische Diagnostik ist in diesen Fällen notwendig und sinnvoll. Der Maturity Onset Diabetes of the Young (MODY) fällt meist durch seine im Vergleich zum Diabetes mellitus Typ 1 mildere Verlaufsform auf und wird mit einer Häufigkeit von 5–10% aller Diabetesformen beziffert. Der MODY Typ 2 wird durch eine Mutation im Glukokinase-Gen hervorgerufen, der MODY Typ 3 durch eine Mutation im HNF-1α-Gen. Der mitochondriale Diabetes mellitus wird aufgrund der häufig auftretenden Schwerhörigkeit auch als MIDD (Maternally Inherited Diabetes and Deafness) bezeichnet und durch Mutationen im mitochondrialen Genom hervorgerufen. Weiterhin wurden in den letzten Jahren verschiedene Genmutationen beim sehr seltenen neonatalen Diabetes mellitus (transienter und permanenter neonataler Diabetes mellitus) aufgeklärt.

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