Difference in stability of the N-domain underlies distinct intracellular properties of the E1064A and H1069Q mutants of copper-transporting ATPase ATP7B

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 Glu(1064) and His(1069) 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.     

Intracellular mislocalization of mutant podocin and correction by chemical chaperones

The NPHS2 gene encoding the podocin protein was causally linked to the autosomal recessive type of steroid-resistant nephrotic syndrome. In this study, we investigated the consequence of the R138Q mutation of podocin, one of the most common missense mutations in the NPHS2 gene, by examining the expression of the wild-type and R138Q mutant podocins in mammalian cells. Either myc- or FLAG-tagged wild-type podocin was strongly stained in plasma membrane, particularly in the fine processes wherein the protein was colocalized with actin stress fibers. On the other hand, the R138Q mutant podocin was completely retained intracellularly and colocalized with the endoplasmic reticulum (ER) marker, calnexin. These results suggest that the R138Q mutation affected podocin protein folding, thereby interfering with the mutant protein's departure from the ER. To determine if the ER retention of R138Q mutant is correctable, cells were incubated with the chemical chaperones glycerol, trimethylamine-N-oxide, and DMSO. Using these two methods, namely, cell surface labeling with sulfo-NHS-S-S-biotin and Alexa 488-streptavidin, and immunostaining to detect the podocin protein close to the plasma membrane, we confirmed that these chemical chaperone treatments elicit a cellular redistribution of R138Q podocin. Our results reveal defective cellular processing of the mutant podocin, and provide evidence for pharmacological correction of the processing defect.     

Stability and ATP binding of the nucleotide-binding domain of the Wilson disease protein: effect of the common H1069Q mutation

Perturbation of the human copper-transporter Wilson disease protein (ATP7B) causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the nucleotide-binding subdomain (N-domain), including the most common mutation, H1069Q. To gain insight into the biophysical behavior of the N-domain under normal and disease conditions, we have characterized wild-type and H1069Q recombinant N-domains in vitro and in silico. The mutant has only twofold lower ATP affinity compared to that of the wild-type N-domain. Both proteins unfold in an apparent two-state reaction at 20 °C and ATP stabilizes the folded state. The thermal unfolding reactions are irreversible and, for the same scan rate, the wild-type protein is more resistant to perturbation than the mutant. For both proteins, ATP increases the activation barrier towards thermal denaturation. Molecular dynamics simulations identify specific differences in both ATP orientation and protein structure that can explain the absence of catalytic activity for the mutant N-domain. Taken together, our results provide biophysical characteristics that may be general to N-domains in other P_1B-ATPases as well as identify changes that may be responsible for the H1069Q WD phenotype in vivo.     

From clinical and biochemical to molecular genetic diagnosis of Wilson disease in Latvia

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism characterized by hepatic and/or neurological damage. More than 300 mutations in gene ATP7B causing this defect have been reported. The data on correlation between WD patient genotypes and clinical presentation are controversial. In this paper, the results of ATP7B mutation analysis by testing for mutation H1069Q and direct sequencing of six exons together with the clinical data of 40 Latvian WD patients are presented. Two previously described and two novel mutations as well as one previously reported polymorphism were identified. The H1069Q mutation was present at 52.5% of the disease alleles. One individual among 157 healthy Latvians was also found to be a mutation H1069Q carrier. The estimated incidence of WD in Latvia is ∼1 in 25600. Wide clinical variability was observed among individuals with the same ATP7B genotype, thus supporting the suggestion that modifying factors play an additional role in the pathogenesis of WD. An algorithm for the diagnosis of WD, including testing for mutation H1069Q, is recommended for the populations where mutation H1069Q accounts for 50% of WD alleles or more. The text was submitted authors English.     

A GABAA receptor mutation linked to human epilepsy (gamma2R43Q) impairs cell surface expression of alphabetagamma receptors

A mutation in the gamma2 subunit of the gamma-aminobutyric acid (GABA) type A receptor (GABAR), which changes an arginine to a glutamine at position 43 (R43Q), is linked to familial idiopathic epilepsies. We used radioligand binding, immunoblotting, and immunofluorescence techniques to examine the properties of wild-type alpha1beta2gamma2 and mutant alpha1beta2gamma2R43Q GABARs expressed in HEK 293 cells. The gamma2R43Q mutation had no affect on the binding affinity of the benzodiazepine flunitrazepam. However, in cells expressing alpha1beta2gamma2R43Q GABARs, the number of binding sites for [3H]flunitrazepam relative to wild-type receptors was decreased 75%. Using surface protein biotinylation, affinity purification, and immunoblotting, we demonstrated that expression of cell surface alpha1beta2gamma2R43Q GABARs was decreased. Surface immunostaining of HEK 293 cells expressing alpha1beta2gamma2R43Q GABARs confirmed that surface expression of the gamma2R43Q subunit was reduced. These data demonstrate that the gamma2R43Q mutation impairs expression of cell surface GABARs. A deficit in surface GABAR expression would reduce synaptic inhibition and result in neuronal hyperexcitability, which could explain why families possessing the gamma2R43Q subunit have epilepsy.     

Identification of a novel inactivating R465Q mutation of the calcium-sensing receptor

In this study, we describe a 52-year-old woman, who was diagnosed with familial benign hypocalciuric hypercalcemia (FBHH), a condition characterized by hypercalcemia, low urinary calcium excretion, and normal parathyroid hormone PTH levels, resulting from inactivating mutations of the calcium-sensing receptor (CaSR). In order to identify and characterize the underlying mutation in the CASR gene, direct sequence analysis of CASR exons 2-7 was performed, and functional activity was examined by transient transfection of human embryonic kidney (HEK-293) cells with wild-type and mutant CaSRs, followed by intracellular calcium measurement using fluorometry, and Western blot analysis. Sequence analysis demonstrated, in addition to the already described A986S polymorphism, a novel heterozygous G--> A substitution in CASR exon 5 that causes an arginine to glutamine substitution at codon 465 (R465Q). Functional analysis showed a rightward shift of the dose-response curve with a significant increase of the EC50 from 5.4 mM of the CaSR carrying the A986S polymorphism alone to 11.3 mM of the CaSR carrying the R465Q mutation in the presence of the A986S polymorphism. Western blot analysis of membrane protein revealed an even higher expression level of the R465Q mutant protein compared to wild-type CaSR. In conclusion, we identified a novel heterozygous loss-of-function R465Q mutation of the CASR gene, which is characterized by a blunted response to calcium stimulation, thereby causing FBHH.     

Cross-talk between carboxypeptidase M and the kinin B1 receptor mediates a new mode of G protein-coupled receptor signaling

G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys(9)-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase.     

Characterization of the molecular defect in the ATP7B gene in Wilson disease patients from Yugoslavia

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism resulting from the absence or dysfunction of a copper transporting P-type ATPase (ATP7B). Approximately 150 mutations of the ATP7B have been identified to date. In this paper, we report the results of molecular characterization and genotype-phenotype analysis, which we have carried out on 35 patients from Yugoslavia affected by WD. Using single-strand conformational polymorphism (SSCP) followed by direct sequencing, we characterized the molecular defect in 80% of WD chromosomes and found 11 different mutations, three of which are novel. The most common mutations that accounted for the molecular defect in 71.3% of WD chromosomes were H1069Q (48.9%), 2304-2305insC (11.4%), R616Q (5.7%), and A1003T (5.7%). The results produced in this paper indicate that the best strategy for mutation detection in Yugoslavian patients with WD is an SSCP analysis of exons 14, 8, 5, and 13, where most of the defects (73.1%) lie, followed by mutation analysis of the remaining exons in ATP7B in patients in whom the mutation was not detected by the finitial screening. These data can be used to develop straightforward genetic testing in this population or in other countries composed of a genetically mixed population like the United States, where a significant number of immigrants came from Central and Eastern Europe.     

Polymorphism in exon 10 of the human coagulation factor V gene in a population at risk for sickle cell disease

In Caucasians, the R506Q mutation in exon 10 of the factor V gene (FV Leiden) confers an increased risk of thromboembolism. We have scanned this region of the gene for possible mutations in 450 subjects from populations at risk for sickle cell disease (SCD). The R506Q mutation was absent in subjects from sub-Saharan Africa, whereas its allelic frequency was 2.5% in the West Indies. Only one other substitution with no functional consequences in vitro (R485K) was found (32.4% allelic frequency) in sub-Saharan Africa. Thus, we found no mutations in exon 10 of the FV gene constituting an additional risk factor for thrombosis in SCD in sub-Saharan Africa. This suggests that the putative selective advantage conferred by R506Q does not exist in these populations, unless R485K has functional consequences in vivo. It further suggests that R506Q in American Africans is of Caucasian origin. Our data are the first to document ethnic variations in the frequency of the R485K polymorphism. Received: 16 December 1996 / Accepted: 16 March 1997     

Phenotypic behavior of caveolin-3 R26Q,a mutant associated with hyperCKemia,distal myopathy,and rippling muscle disease

Four different phenotypes have been associated with CAV3 mutations: limb girdle muscular dystrophy-1C (LGMD-1C), rippling muscle disease (RMD), and distal myopathy (DM), as well as idiopathic and familial hyperCKemia (HCK). Detailed molecular characterization of two caveolin-3 mutations (P104L and TFT), associated with LGMD-1C, shows them to impart a dominant-negative effect on wild-type caveolin-3, rendering it dysfunctional through sequestration in the Golgi complex. Interestingly, substitution of glutamine for arginine at amino acid position 26 (R26Q) of caveolin-3 is associated not only with RMD but also with DM and HCK. However, the phenotypic behavior of the caveolin-3 R26Q mutation has never been evaluated in cultured cells. Thus we characterized the cellular and molecular properties of the R26Q mutant protein to better understand how this mutation can manifest as such distinct disease phenotypes. Here, we show that the caveolin-3 R26Q mutant is mostly retained at the level of the Golgi complex. The caveolin-3 R26Q mutant formed oligomers of a much larger size than wild-type caveolin-3 and was excluded from caveolae-enriched membranes. However, caveolin-3 R26Q did not behave in a dominant-negative fashion when coexpressed with wild-type caveolin-3. Thus the R26Q mutation behaves differently from other caveolin-3 mutations (P104L and TFT) that have been previously characterized. These data provide a possible explanation for the scope of the various disease phenotypes associated with the caveolin-3 R26Q mutation. We propose a haploinsufficiency model in which reduced levels of wild-type caveolin-3, although not rendered dysfunctional due to the caveolin-3 R26Q mutant protein, are insufficient for normal muscle cell function. muscle cell caveolae; caveolin-3; muscular dystrophy     

Mutational analysis of the Menkes copper P-type ATPase (ATP7A)

The Menkes protein (ATP7A; MNK) is a ubiquitous human copper-translocating P-type ATPase and it has a key role in regulating copper homeostasis. Previously we characterised fundamental steps in the catalytic cycle of the Menkes protein. In this study we analysed the role of several conserved regions of the Menkes protein, particularly within the putative cytosolic ATP-binding domain. The results of catalytic studies have indicated an important role of 1086His in catalysis. Our findings provide a biochemical explanation for the most common Wilson disease-causing mutation (H1069Q in the homologous Wilson copper-translocating P-type ATPase). Furthermore, we have identified a unique role of 1230Asp, within the DxxK motif, in coupling ATP binding and acylphosphorylation with copper translocation. Finally, we found that the Menkes protein mutants with significantly reduced catalytic activity can still undergo copper-regulated exocytosis, suggesting that only the complete loss of catalytic activity prevents copper-regulated trafficking of the Menkes protein.     

A Multiplex Human Syndrome Implicates a Key Role for Intestinal Cell Kinase in Development of Central Nervous, Skeletal, and Endocrine Systems

Six infants in an Old Order Amish pedigree were observed to be affected with endocrine-cerebro-osteodysplasia (ECO). ECO is a previously unidentified neonatal lethal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal systems. Autozygosity mapping and sequencing identified a previously unknown missense mutation, R272Q, in ICK, encoding intestinal cell kinase (ICK). Our results established that R272 is conserved across species and among ethnicities, and three-dimensional analysis of the protein structure suggests protein instability due to the R272Q mutation. We also demonstrate that the R272Q mutant fails to localize at the nucleus and has diminished kinase activity. These findings suggest that ICK plays a key role in the development of multiple organ systems.     

The role of the invariant His-1069 in folding and function of the Wilson's disease protein,the human copper-transporting ATPase ATP7B

The copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in ATP7B lead to Wilson's disease, a severe disorder with neurological and hepatic manifestations. One of the most common disease mutations, a H1069Q substitution, causes intracellular mislocalization of ATP7B (the Wilson's disease protein, WNDP). His-1069 is located in the nucleotide-binding domain of WNDP and is conserved in all copper-transporting ATPases from bacteria to mammals; however, the specific role of this His in the structure and function of WNDP remains unclear. We demonstrate that substitution of His-1069 for Gln, Ala, or Cys does not significantly alter the folding of the WNDP nucleotide-binding domain or the proteolytic resistance of the full-length WNDP. In contrast, the function of WNDP is markedly affected by the mutations. The ability to form an acylphosphate intermediate in the presence of ATP is entirely lost in all three mutants, suggesting that His-1069 is important for ATP-dependent phosphorylation. Other steps of the WNDP enzymatic cycle are less dependent on His-1069. The H1069C mutant shows normal phosphorylation in the presence of inorganic phosphate; it binds an ATP analogue, beta,gamma-imidoadenosine 5'-triphosphate (AMP-PNP), and copper and undergoes nucleotide-dependent conformational transitions similar to those of the wild-type WNDP. Although binding of AMP-PNP is not disrupted by the mutation, the apparent affinity for the nucleotide is decreased by 4-fold. We conclude that His-1069 is responsible for proper orientation of ATP in the catalytic site of WNDP prior to ATP hydrolysis.     

A point mutation uncouples transducin-alpha from the photoreceptor RGS and effector proteins

A novel gain-of-function mutation, R243Q, has been recently identified in the Candida elegans Gqalpha protein EGL-30. The position corresponding to Arg243 in EGL-30 is absolutely conserved among heterotrimeric G proteins. This mutation appears to be the first gain-of-function mutation in the switch III region of Galpha subunits. To investigate consequences of the R-->Q mutation we introduced the corresponding R238Q mutation into transducin-like Gtalpha* subunit. The mutant retained intact interactions with Gtbetagamma and rhodopsin but exhibited a twofold reduction in the kcat value for guanosine 5'-triphosphate (GTP) hydrolysis. The GTPase activity of R238Q was not accelerated by the RGS domain of the visual GTPase-activating protein, RGS9-1. In addition, R238Q displayed a significant impairment in the effector function. Our data and the crystal structures of transducin suggest that the major reason for the reduced intrinsic GTPase activity of R238Q and the lack of RGS9 function is the break of the conserved ionic contact between Arg238 and Glu39, which apparently stabilizes the transitional state for GTP hydrolysis. We hypothesize that the R243Q mutation in EGL-30 severs the ionic interaction of Arg243 with Glu43, leading to a defective inactivation of the mutant by the C. elegans RGS protein EAT-16.     

Characterization of a mutant R11H αB-crystallin associated with human inherited cataract

αB-Crystallin plays an important part in cataract development. A novel mutation (R11H) was previously detected by our group. In the present study, we set out to investigate the possible molecular mechanism by which the R11H mutation causes cataract. We found that the mutant αB-crystallin exhibits folding defects, decreased surface hydrophobicity and enhanced chaperone-like activity compared with the wild-type αB-crystallin. The mutant protein shows nearly the same molecular mass and thermal stability as the wild-type form. Transfection studies revealed that the R11H mutant was remarkably similar to the wild-type protein in its subcellular distribution, but has an abnormal ability to induce cell apoptosis. These results suggest that the changes in hydrophobic exposure and the abnormal ability to induce programmed cell death of the mutant protein are likely to be responsible for the onset of cataract.     

Analysis of most common mutations R778G, R778L, R778W, I1102T and H1069Q in Indian Wilson disease patients: Correlation between genotype/phenotype/copper ATPase activity

The present study was intented to estimate the frequencies of the most common mutations (R778L, R778W, R778G, I1102T and H1069Q) of ATP7B in Indian Wilson disease (WD) population and to explore the correlation between genotype/phenotype and copper ATPase activity. A total of 33 WD patients and their family members from North West states of India were examined. The H1069Q, R778W and R778L mutations were absent in these WD patients. R778W and I1102T mutations were present in 36% of WD patients. Family analysis for these mutations using PCR-RFLP documented 5 carriers and 2 asymptomatic WD patients. The copper ATPase activity in WD patients was significantly reduced (50%) than that of control individuals. No significant difference was observed in copper stimulated ATPase activity between homozygous (R778W/R778W, I1102T/I1102T) and compound heterozygous (R778W/unknown mutation, I1102T/unknown mutation) WD patients. Serum ceruloplasmin, serum copper levels were significantly lower in homozygous WD patients than that of compound heterozygous. However, no significant difference was observed in liver copper contents between heterozygous and homozygous patients. In conclusion, the data suggest that R778W and I1102T are most common mutations and provide the basis of genetic (PCR-RFLP) diagnostic tool for Indian WD patients as well as in siblings/parents where biochemical parameters are ambiguous.