guanosine-triphosphate and Syndrome

Hyperinsulinism/hyperammonemia (HI/HA) syndrome has been known to be caused by dominant gain-of-function mutations in GLUD1, encoding the mitochondrial enzyme glutamate dehydrogenase. Pathogenic GLUD1 mutations enhance enzymatic activity by reducing its sensitivity to allosteric inhibition by GTP. Two recent independent studies showed that a similar HI/HA phenotype can be caused by biallelic mutations in SLC25A36, encoding pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine and guanine nucleotides across the inner mitochondrial membrane: one study reported a single case caused by a homozygous truncating mutation in SLC25A36 resulting in lack of expression of SLC25A36 in patients' fibroblasts. A second study described two siblings with a splice site mutation in SLC25A36, causing reduction of mitochondrial GTP content, putatively leading to hyperactivation of glutamate dehydrogenase. In an independent study, through combined linkage analysis and exome sequencing, we demonstrate in four individuals of two Bedouin Israeli related families the same disease-causing SLC25A36 (NM_018155.3) c.284 + 3A > T homozygous splice-site mutation found in the two siblings. We demonstrate that the mutation, while causing skipping of exon 3, does not abrogate expression of mRNA and protein of the mutant SLC25A36 in patients' blood and fibroblasts. Affected individuals had hyperinsulinism, hyperammonemia, borderline low birth weight, tonic-clonic seizures commencing around 6 months of age, yet normal intellect and no significant other morbidities. Chronic constipation, hypothyroidism, and developmental delay previously described in a single patient were not found. We thus verify that biallelic SLC25A36 mutations indeed cause HI/HA syndrome and clearly delineate the disease phenotype.

Topics: Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperammonemia; Hyperinsulinism; Mitochondrial Membrane Transport Proteins; Mutation; Syndrome

Mitochondrial GTP (mtGTP)-insensitive mutations in glutamate dehydrogenase (GDH(H454Y)) result in fasting and amino acid-induced hypoglycemia in hyperinsulinemia hyperammonemia (HI/HA). Surprisingly, hypoglycemia may occur in this disorder despite appropriately suppressed insulin. To better understand the islet-specific contribution, transgenic mice expressing the human activating mutation in β-cells (H454Y mice) were characterized in vivo. As in the humans with HI/HA, H454Y mice had fasting hypoglycemia, but plasma insulin concentrations were similar to the controls. Paradoxically, both glucose- and glutamine-stimulated insulin secretion were severely impaired in H454Y mice. Instead, lack of a glucagon response during hypoglycemic clamps identified impaired counterregulation. Moreover, both insulin and glucagon secretion were impaired in perifused islets. Acute pharmacologic inhibition of GDH restored both insulin and glucagon secretion and normalized glucose tolerance in vivo. These studies support the presence of an mtGTP-dependent signal generated via β-cell GDH that inhibits α-cells. As such, in children with activating GDH mutations of HI/HA, this insulin-independent glucagon suppression may contribute importantly to symptomatic hypoglycemia. The identification of a human mutation causing congenital hypoglucagonemic hypoglycemia highlights a central role of the mtGTP-GDH-glucagon axis in glucose homeostasis.

Topics: Amino Acids; Animals; Glucagon; Glucagon-Secreting Cells; Glucose Clamp Technique; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperammonemia; Hyperinsulinism; Hypoglycemia; Insulin; Insulin Secretion; Insulin-Secreting Cells; Mice; Mice, Transgenic; Mitochondria; Mutation; Syndrome

Costello syndrome (CS) is a rare congenital disorder characterized by failure to thrive, craniofacial dysmorphisms, cardiac and skin abnormalities, mental retardation, and predisposition to malignancies. CS is caused by heterozygous gain-of-function mutations in HRAS that also occur as somatic alterations in human tumors. HRAS is one of the three classical RAS proteins and cycles between an active, GTP- and an inactive, GDP-bound conformation. We used primary human skin fibroblasts from patients with CS as a model system to study the functional consequences of HRAS mutations on endogenous signaling pathways. The GTP-bound form of HRAS was significantly enriched in CS compared with normal fibroblasts. Active HRAS is known to stimulate both the RAF-MEK-ERK and the PI3K-AKT signaling cascade. Phosphorylation of MEK and ERK was normal in CS fibroblasts under basal conditions and slightly prolonged after epidermal growth factor (EGF) stimulation. Interestingly, basal phosphorylation of AKT was increased yet more in CS fibroblasts. Moreover, AKT phosphorylation was diminished in the early and enhanced in the late phase of EGF stimulation. Taken together, these results document that CS-associated HRAS mutations result in prolonged signal flux in a ligand-dependent manner. Our data suggest that altered cellular response to growth factors rather than constitutive activation of HRAS downstream signaling molecules may contribute to some of the clinical features in patients with CS.

Topics: Abnormalities, Multiple; Cells, Cultured; Chromones; Craniofacial Abnormalities; Enzyme Inhibitors; Epidermal Growth Factor; Failure to Thrive; Fibroblasts; Guanosine Triphosphate; Humans; Immunoblotting; Intellectual Disability; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Morpholines; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction; Skin; Skin Abnormalities; Syndrome

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with beta1 integrin, alpha-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.

Topics: Abnormalities, Multiple; Adult; Cell Line, Transformed; Cell Movement; Cell Shape; Child; Enzyme Activation; Focal Adhesions; Guanosine Triphosphate; Humans; Keratinocytes; Membrane Proteins; Middle Aged; Models, Biological; Mucous Membrane; Neoplasm Proteins; Phenotype; Phosphorylation; Protein Binding; Pseudopodia; rho GTP-Binding Proteins; RNA, Small Interfering; Skin Abnormalities; Syndrome

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the alpha 4 (Ala152Pro) or beta 5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.

Topics: Albinism; Amino Acid Substitution; Animals; Cell Degranulation; Cell Line; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Immunologic Deficiency Syndromes; Melanocytes; Melanosomes; Mice; Mutation; Protein Binding; rab GTP-Binding Proteins; rab27 GTP-Binding Proteins; Syndrome

Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blotting, Western; Carrier Proteins; Fluorescent Antibody Technique; Guanosine Triphosphate; Humans; Immunologic Deficiency Syndromes; Mice; Mutation, Missense; Myosin Heavy Chains; Myosin Type V; Nervous System Diseases; Protein Binding; rab GTP-Binding Proteins; rab27 GTP-Binding Proteins; Syndrome; Tumor Cells, Cultured

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism in which affected children have recurrent symptomatic hypoglycemia together with asymptomatic, persistent elevations of plasma ammonium levels. We have shown that the disorder is caused by dominant mutations of the mitochondrial enzyme, glutamate dehydrogenase (GDH), that impair sensitivity to the allosteric inhibitor, GTP. In 65 HI/HA probands screened for GDH mutations, we identified 19 (29%) who had mutations in a new domain, encoded by exons 6 and 7. Six new mutations were found: Ser(217)Cys, Arg(221)Cys, Arg(265)Thr, Tyr(266)Cys, Arg(269)Cys, and Arg(269)HIS: In all five mutations tested, lymphoblast GDH showed reduced sensitivity to allosteric inhibition by GTP (IC(50), 60--250 vs. 20--50 nmol/L in normal subjects), consistent with a gain of enzyme function. Studies of ATP allosteric effects on GDH showed a triphasic response with a decrease in high affinity inhibition of enzyme activity in HI/HA lymphoblasts. All of the residues altered by exons 6 and 7 HI/HA mutations lie in the GTP-binding domain of the enzyme. These data confirm the importance of allosteric regulation of GDH as a control site for amino acid-stimulated insulin secretion and indicate that the GTP-binding site is essential for regulation of GDH activity by both GTP and ATP.

Topics: Enzyme Inhibitors; Exons; Female; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperammonemia; Hyperinsulinism; Infant; Male; Mutation; Polymorphism, Genetic; Protein Structure, Tertiary; Syndrome

Hyperinsulinism and hyperammonemia syndrome has been reported as a cause of moderately severe hyperinsulinism with diffuse involvement of the pancreas. The disorder is caused by gain of function mutations in the GLUD1 gene, resulting in a decreased inhibitory effect of guanosine triphosphate on the glutamate dehydrogenase (GDH) enzyme. Twelve unrelated patients (six males, six females) with hyperinsulinism and hyperammonemia syndrome have been investigated. The phenotypes were clinically heterogeneous, with neonatal and infancy-onset hypoglycemia and variable responsiveness to medical (diazoxide) and dietary (leucine-restricted diet) treatment. Hyperammonemia (90-200 micromol/L, normal <50 micromol/L) was constant and not influenced by oral protein, by protein- and leucine-restricted diet, or by sodium benzoate or N-carbamylglutamate administration. The patients had mean basal GDH activity (18.3 +/- 0.9 nmol/min/mg protein) not different from controls (17.9 +/- 1.8 nmol/min/mg protein) in cultured lymphoblasts. The sensitivity of GDH activity to inhibition by guanosine triphosphate was reduced in all patient lymphoblast cultures (IC(50), or concentrations required for 50% inhibition of GDH activity, ranging from 140 to 580 nM, compared with control IC(50) value of 83 +/- 1.0 nmol/L). The allosteric effect of ADP was within the normal range. The activating effect of leucine on GDH activity varied among the patients, with a significant decrease of sensitivity that was correlated with the negative clinical response to a leucine-restricted diet in plasma glucose levels in four patients. Molecular studies were performed in 11 patients. Heterozygous mutations were localized in the antenna region (four patients in exon 11, two patients in exon 12) as well as in the guanosine triphosphate binding site (two patients in exon 6, two patients in exon 7) of the GLUD1 gene. No mutation has been found in one patient after sequencing the exons 5-13 of the gene.

Topics: Adolescent; Blood Glucose; Child; Child, Preschool; Diet; Female; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperammonemia; Hyperinsulinism; Infant; Infant, Newborn; Leucine; Lymphocytes; Male; Syndrome

Glutamate dehydrogenase (GDH) is allosterically activated by the amino acid leucine to mediate protein stimulation of insulin secretion. Children with the hyperinsulinism/hyperammonemia (HI/HA) syndrome have symptomatic hypoglycemia plus persistent elevations of plasma ammonium. We have reported that HI/HA may be caused by dominant mutations of GDH that lie in a unique allosteric domain that is encoded within GDH exons 11 and 12. To examine the frequency of mutations in this domain, we screened genomic DNA from 48 unrelated cases with the HI/HA syndrome for exon 11 and 12 mutations in GDH. Twenty-five (52%) had mutations in these exons; 74% of the mutations were sporadic. Clinical manifestations included normal birth weight, late onset of hypoglycemia, diazoxide responsiveness, and protein-sensitive hypoglycemia. Enzymatic studies of lymphoblast GDH in seven of the mutations showed that all had reduced sensitivity to inhibition with GTP, consistent with an increase in enzyme activity. Mutations had little or no effect on enzyme responses to positive allosteric effectors, such as ADP or leucine. Based on the three-dimensional structure of GDH, the mutations may function by impairing the binding of an inhibitory GTP to a domain responsible for the allosteric and cooperativity properties of GDH.

Topics: Adenosine Diphosphate; Allosteric Site; Amino Acid Sequence; Ammonia; Enzyme Activation; Exons; Female; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperinsulinism; Infant; Infant, Newborn; Lymphocytes; Male; Models, Molecular; Molecular Sequence Data; Mutation; Syndrome

Hyperinsulinism-hyperammonemia syndrome (HHS) is a recently identified genetic disorder characterized by hyperinsulinemic hypoglycemia with concomitant hyperammonemia. In patients with HHS, activating mutations in the glutamate dehydrogenase (GDH) gene have been identified. GDH is a key enzyme linking glutamate metabolism with the Krebs cycle and catalyzes the conversion of glutamate to alpha-ketoglutarate. The activity of GDH is controlled by allosteric inhibition by GTP and, so far, all the mutations of HHS patients have been located within the GTP-binding site. Characteristically, GDH from these individuals have therefore normal basal activity in conjunction with a loss of GTP inhibition. In this study, however, we have identified a novel variant GDH in a patient with a more severe form of HHS. The mutation is located outside the GTP-binding site and the patient's GDH shows consistently higher activity, even in the absence of allosteric effectors. These results further support the hypothesis that the activating mutation of GDH is the cause of HHS. The mechanism leading to the activation of GDH, however, is not always related to the loss of GTP inhibition as was originally suggested.

Topics: Adenosine Diphosphate; Ammonia; Base Sequence; Dose-Response Relationship, Drug; Genetic Markers; Genotype; Glutamate Dehydrogenase; Guanosine Triphosphate; Humans; Hyperinsulinism; Kinetics; Male; Metabolism, Inborn Errors; Molecular Sequence Data; Point Mutation; Polymorphism, Genetic; Syndrome

A syndrome of hyperuricemia, sensorineural deafness, mild mental handicap and congenital disequilibrium in a four-year-old boy is probably inherited as a sex-linked condition since his mother has sensorineural deafness and similar biochemical abnormalities. There is evidence of a superactive PP-ribose-P synthetase, normal purine salvage enzymes, and severe depletion of nicotinamide adenine dinucleotide and guanine triphosphate in red cells.

Topics: Deafness; Erythrocytes; Female; Guanosine Triphosphate; Humans; Infant; Infant, Newborn; Intellectual Disability; Male; NAD; Purine-Pyrimidine Metabolism, Inborn Errors; Ribose-Phosphate Pyrophosphokinase; Syndrome; Uric Acid; X Chromosome