Disease-associated GPR56 mutations cause bilateral frontoparietal polymicrogyria via multiple mechanisms

Loss-of-function mutations in the gene encoding G protein-coupled receptor 56 (GPR56) lead to bilateral frontoparietal polymicrogyria (BFPP), an autosomal recessive disorder affecting brain development. The GPR56 receptor is a member of the adhesion-GPCR family characterized by the chimeric composition of a long ectodomain (ECD), a GPCR proteolysis site (GPS), and a seven-pass transmembrane (7TM) moiety. Interestingly, all identified BFPP-associated missense mutations are located within the extracellular region of GPR56 including the ECD, GPS, and the extracellular loops of 7TM. In the present study, a detailed molecular and functional analysis of the wild-type GPR56 and BFPP-associated point mutants shows that individual GPR56 mutants most likely cause BFPP via different combination of multiple mechanisms. These include reduced surface receptor expression, loss of GPS proteolysis, reduced receptor shedding, inability to interact with a novel protein ligand, and differential distribution of the 7TM moiety in lipid rafts. These results provide novel insights into the cellular functions of GPR56 receptor and reveal molecular mechanisms whereby GPR56 mutations induce BFPP.     

Disease-associated mutations inactivate AMP-lysine hydrolase activity of Aprataxin

Ataxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead-associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage, but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein NH2-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site-dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity, while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations.     

Biochemical characterization of genetic mutations of GPR56 in patients with bilateral frontoparietal polymicrogyria (BFPP)

Bilateral frontoparietal polymicrogyria (BFPP) is a rare genetic disease characterized by cortical malformation associated with GPR56 mutations of frameshift, splicing, and point mutations (Science 303:2033). All the missense point mutations are located in the regions predicted to be exposed at the cell surface, e.g. the N-terminal extracellular domain (ECD), the proteolytic site (GPS), and the extracellular loops of transmembrane domain (TM), implying functionally important interaction among these domains. Wild type GPR56 protein is cleaved at the GPCR protein cleavage site (GPS) and gives rise to two subunits (ECD and TM), which are transported to cell surface. We have shown that GPR56 GPS mutant protein is defective in cleavage and surface localization, while non-GPS mutant proteins are cleaved normally but still defective in surface localization. Furthermore, all the mutant proteins demonstrated different glycosylation pattern from that of wild-type protein. PNGase F and Endo H sensitivity assays suggests that the mutant proteins are trapped in endoplasmic reticulum (ER), preventing them from trafficking to Golgi where further glycosylation modification usually occurs before destination to cell surface. Therefore, the loss-of-function of all these missense mutations is primarily caused by their failure to localize to cell surface.     

Disease-associated N-terminal complement factor H mutations perturb cofactor and decay-accelerating activities

Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19-20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1-4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1-4(V62) and FH1-4(I62) bind immobilized C3b with similar affinities (K(D) = 10-14 μM), whereas FH1-4(I62) is slightly more effective than FH1-4(V62) as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1-4(V62) binds to C3b with comparable affinity (K(D) ～12 μM) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1-4(V62), binds poorly to immobilized C3b (K(D) >35 μM) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.     

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer–dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.     

Disease-associated mutations in SOD1 are impervious to dominant positive or negative effects

The familial form of amyotrophic lateral sclerosis is caused by mutations in the SOD1 gene encoding the cytosolic antioxidant enzyme Cu,Zn superoxide dismutase. Although there is no clear correlation between disease and dismutating catalytic activity among the various disease-associated SOD1 alleles, all of the known missense mutations significantly alter the half-life of the encoded polypeptides. Using transient transfection studies in mammalian cells, it was demonstrated that a frameshift mutation in SOD1 which results in a truncated polypeptide is similarly destabilized. Using an epitope-tagging strategy to discriminate between mutant and wild-type SOD1 polypeptides, no evidence for dominant effects on polypeptide stability was detected, including that of a positive effect of the wild-type on mutant SOD1 polypeptides or that of a negative effect of mutant on wild-type SOD1 polypeptides. These experiments thus favor a non-catalytic role of mutant forms of SOD1 in disease progression.     

Disease-associated mutations in human mannose-binding lectin compromise oligomerization and activity of the final protein

Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the biological consequences of MBL mutations, we expressed wild type MBL and mutated MBL in Chinese hamster ovary cells. The normal MBL cDNA (WT MBL-A) was cloned, and the three known natural and two artificial variants were expressed in Chinese hamster ovary cells. When analyzed, WT MBL-A formed covalently linked higher oligomers with a molecular mass of about 300-450 kDa, corresponding to 12-18 single chains or 4-6 structural units. By contrast, all MBL variants formed a dominant band of about 50 kDa, with increasingly weaker bands at 75, 100, and 125 kDa corresponding to two, three, four, and five chains, respectively. In contrast to WT MBL-A, variant MBL formed noncovalent oligomers containing up to six chains (two structural units). MBL variants bound ligands with a markedly reduced capacity compared with WT MBL-A. Mutations in the collagenous region of human MBL compromise assembly of higher order oligomers, resulting in reduced ligand binding capacity and thus reduced capability to activate complement.     

Disease-associated mutations and alternative splicing alter the enzymatic and motile activity of nonmuscle myosins II-B and II-C

Human families with single amino acid mutations in nonmuscle myosin heavy chain (NMHC) II-A (MYH9) and II-C (MYH14) have been described as have mice generated with a point mutation in NMHC II-B (MYH10). These mutations (R702C and N93K in human NMHC II-A, R709C in murine NMHC II-B, and R726S in human NMHC II-C) result in phenotypes affecting kidneys, platelets, and leukocytes (II-A), heart and brain (II-B), and the inner ear (II-C). To better understand the mechanisms underlying these defects, we characterized the in vitro activity of mutated and wild-type baculovirus-expressed heavy meromyosin (HMM) II-B and II-C. We also expressed two alternatively spliced isoforms of NMHC II-C which differ by inclusion/exclusion of eight amino acids in loop 1, with and without mutations. Comparison of the actin-activated MgATPase activity and in vitro motility shows that mutation of residues Asn-97 and Arg-709 in HMM II-B and the homologous residue Arg-722 (Arg-730 in the alternatively spliced isoform) in HMM II-C decreases both parameters but affects in vitro motility more severely. Analysis of the transient kinetics of the HMM II-B R709C mutant shows an extremely tight affinity of HMM for ADP and a very slow release of ADP from acto-HMM. Although mutations generally decreased HMM activity, the R730S mutation in HMM II-C, unlike the R730C mutation, had no effect on actin-activated MgATPase activity but decreased the rate of in vitro motility by 75% compared with wild type. Insertion of eight amino acids into the HMM II-C heavy chain increases both actin-activated MgATPase activity and in vitro motility.     

Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response

Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNA^Glu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics.     

The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of β-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with β-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.     

GPR56 and the Developing Cerebral Cortex: Cells, Matrix, and Neuronal Migration

GPR56, a member of the adhesion G protein-coupled receptor (GPCR) family, is integral to the development of the cortex, as mutations in GPR56 cause bilateral frontoparietal polymicrogyria (BFPP). BFPP is a cobblestone-like cortical malformation, characterized by overmigrating neurons and the formation of neuronal ectopias on the surface of the brain. Since its original cloning a decade ago, GPR56 has emerged from an orphaned and uncharacterized protein to an increasingly well-understood receptor, both in terms of its signaling and function. Collagen III is the ligand of GPR56 in the developing brain. Upon binding to collagen III, GPR56 activates RhoA via coupling to Gα_12/13. This pathway appears to be particularly critical in the preplate neurons, which are the earliest born neurons in the cortex, as the expression pattern of GPR56 in these neurons mimics the anterior to posterior gradient of malformation associated with loss of GPR56 in both humans and mice. Further characterizing the role of GPR56 in the preplate will shed light on the mechanism of cortical development and patterning.     

Holoenzyme DNA polymerase III fixes mutations

DNA polymerase III is required for mutagenesis after damage to the chromosome. This effect is not modulated by the presence or absence of DNA polymerase II activity in the cell. In cells containing a temperature-sensitive dnaE mutation, the alpha-subunit of DNA polymerase III is inactivated at the restrictive temperature, resulting in lethality. Cells containing the pcbA1 mutation can continue replication if DNA polymerase I activity is present. When such cells are shifted from the permissive to the restrictive temperature, mutagenesis decreases rapidly after 10 min. These results are compatible with conversion of the replicative apparatus from one containing a functional DNA polymerase III synthetic subunit to one containing DNA polymerase I. We also find that DNA polymerase I dependent replication is markedly sensitive to coumermycin A1. We conclude that DNA polymerase III holoenzyme with the alpha-subunit is required for fixing mutations in the genome.     

GPR56 and TG2: Possible Roles in Suppression of Tumor Growth by the Microenvironment

Metastasis is a complex process that involves multiple levels of cell-cell interaction. Among these interactions, tumor-stroma interactions are being actively investigated. Metastatic cells are hypothesized to show gene expression changes that contribute to their survival and growth at the distant site. Such chances could contribute either to enhancement of growth or to evasion of growth inhibition by the normal tissue environment thus allowing growth as metastases. Our recent report that tumors from highly metastatic melanoma derivatives express low levels of a suppressor of tumor progression, GPR56, is consistent with such a model. GPR56 associates in a complex with Gαq and the tetraspanin CD81. We further identified a ligand that interacts with GPR56 in the extracellular matrix (ECM) as TG2, a major crosslinking enzyme in the matrix. TG2 also binds to fibronectin and integrins and affects their cell adhesion functions. TG2 itself has been implicated in suppression of tumor progression; therefore TG2 might serve as a host defense against the invading metastatic cells. The highly metastatic cells may escape from this inhibition by down-regulation of GPR56. Much future work will be needed to test this hypothesis and further our understanding of metastasis in general.     

Disease-associated mutations at copper ligand histidine residues of superoxide dismutase 1 diminish the binding of copper and compromise dimer stability

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with (-)ONOO and H(2)O(2) in vitro. This reactivity requires a copper ion bound in the active site and is a suggested mechanism of motor neuron injury. However, we have found that transgenic mice that express SOD1-H46R/H48Q, which combines natural FALS mutations at ligands for copper and which is inactive, develop motor neuron disease. Using a direct radioactive copper incorporation assay in transfected cells and the established tools of single crystal x-ray diffraction, we now demonstrate that this variant does not stably bind copper. We find that single mutations at copper ligands, including H46R, H48Q, and a quadruple mutant H46R/H48Q/H63G/H120G, also diminish the binding of radioactive copper. Further, using native polyacrylamide gel electrophoresis and a yeast two-hybrid assay, the binding of copper was found to be related to the formation of the stable dimeric enzyme. Collectively, our data demonstrate a relationship between copper and assembly of SOD1 into stable dimers and also define disease-causing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry.     

Nucleo-cytoplasmic interaction between oligomycin-resistant mutations in Saccharomyces cerevisiae

1.A single-gene nuclear mutant of Saccharomyces cerevisiae, isolated as oligomycin-resistant, exhibits in vivo cross-resistance to venturicidin and collateral sensitivity to Synthalin. All three compounds are inhibitors of mitochondrial oxidative phosphorylation. Oligomycin resistance and Synthalin sensitivity are recessive, while venturicidin resistance is dominant. 2. Acytoplasmic mutant, also isolated as oligomycin-resistant, shows collateral sensitivity to both Synthalin and venturicidin. All three traits undergo mitotic segregation in diploids formed by crossing mutant and normal halpoids. 3. A novel nucleocytoplasmic interaction is observed in diploids formed by crossing haploid strains containing the nuclear and the cytoplasmic mutations, respectively. The dominant venturicidin resistance determined by the nuclear gene undergoes mitotic segregation, which results from a suppression of the nuclear phenotype by the cytoplasmic mutation. When a diploid mitotic segregant contains primarily mutant-type mitochondria, venturicidin resistance is completely suppressed. In haploids containing both the nuclear and cytoplasmic mutations, suppression is only partial. 4. Oxidative phosphorylation and ATPase in mitochondrial fractions isolated fromcytoplasmic mutant cells are less sensitive to inhibition by oligomycin than normal, but in vitro sensitivity to venturicidin is not significantly changed. In similar mitochondrial fractions isolated from normal and nuclear mutant cells, no significant differences in sensitivity to either inhibitor are detected. 5. The molecular basis for the nucleocytoplasmic suppression of venturicidin resistance may involve participation of mitochondrial membrane, plasma membrane or both. Either mitochondria can undergo changes in venturicidin sensitivity upon isolation, or the molecular entity which controls access of venturicidin to the mitochondria resides outside of the organelles. 6. Our data establish that aspects of the response in vivo of both venturicidin and Snythalin are controlled by the mitochondrial genome. 7. The nucleocytoplasmic interaction described here is the first example in which a specific restricted mitochondrial mutation modifies the phenotypic expression of a nuclear gene.     

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs^∗57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.     

Desmin mutations in the terminal consensus motif prevent synemin-desmin heteropolymer filament assembly

Disorganization of the desmin network is associated with cardiac and skeletal myopathies characterized by accumulation of desmin-containing aggregates in the cells. Multiple associations of intermediate filament proteins form a network to increase mechanical and functional stability. Synemin is a desmin-associated type VI intermediate filament protein. Neither its impact on desmin network nor how it integrates into desmin filament is yet elucidated. To gain more insight into the molecular basis of these processes, we coexpressed synemin with different desmin mutants in ex vivo models. The screening of fourteen desmin mutants showed that synemin with desmin mutants revealed two behaviors. Firstly, synemin was co-localized in desmin aggregates and its coexpression decreased the number of cells containing aggregates. Secondly, synemin was excluded from the aggregates, then synemin had no effect on desmin network organization. Among fourteen desmin mutants, there were only three mutants, p.E401K, p.R406W and p.E413K, in which synemin was not found in aggregates. This behavior was correlated to the abnormal salt-bridges of desmin-dimer as seen in silico constructs. Moreover, desmin constructs in silico and published results in literature have predicted that the salt-bridges absence in the desmin filament building prevent longitudinal annealing and/or radial compaction. These results suggest that the state of desmin-filament assembly is crucial for synemin anchorage and consequently might involve mechanical and functional stability of the cytoskeletal network.     

Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels

ATP-sensitive potassium (KATP) channels couple the metabolic status of a cell to its membrane potential-a property that endows pancreatic beta-cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that KATP channels undergo rapid internalisation from the plasma membrane by clathrin-mediated endocytosis. We present several lines of evidence to demonstrate that endocytosis is mediated by a tyrosine based signal (330YSKF333) located in the carboxy-terminus of Kir6.2 and that SUR1 has no direct role. We show that genetic mutations, Y330C and F333I, which cause permanent neonatal diabetes mellitus, disrupt this motif and abrogate endocytosis of reconstituted mutant channels. The resultant increase in the surface density of KATP channels would predispose beta-cells to hyperpolarise and may account for reduced insulin secretion in these patients. The data imply that endocytosis of KATP channels plays a crucial role in the (patho)-physiology of insulin secretion.