Mutant protein kinase Cgamma found in spinocerebellar ataxia type 14 is susceptible to aggregation and causes cell death

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease characterized by various symptoms including cerebellar ataxia. Recently, several missense mutations in the protein kinase Cgamma (gammaPKC) gene have been found in different SCA14 families. To elucidate how the mutant gammaPKC causes SCA14, we examined the molecular properties of seven mutant (H101Y, G118D, S119P, S119F, Q127R, G128D, and F643L) gammaPKCs fused with green fluorescent protein (gammaPKC-GFP). Wild-type gammaPKC-GFP was expressed ubiquitously in the cytoplasm of CHO cells, whereas mutant gammaPKC-GFP tended to aggregate in the cytoplasm. The insolubility of mutant gammaPKC-GFP to Triton X-100 was increased and correlated with the extent of aggregation. gammaPKC-GFP in the Triton-insoluble fraction was rarely phosphorylated at Thr(514), whereas gammaPKC-GFP in the Triton-soluble fraction was phosphorylated. Furthermore, the stimulation of the P2Y receptor triggered the rapid aggregation of mutant gammaPKC-GFP within 10 min after transient translocation to the plasma membrane. Overexpression of the mutant gammaPKC-GFP caused cell death that was more prominent than wild type. The cytotoxicity was exacerbated in parallel with the expression level of the mutant. These results indicate that SCA14 mutations make gammaPKC form cytoplasmic aggregates, suggesting the involvement of this property in the etiology of SCA14.     

Mutant γPKC that causes spinocerebellar ataxia type 14 upregulates Hsp70, which protects cells from the mutant’s cytotoxicity

Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is misfolded, susceptible to aggregation and cytotoxic. Molecular chaperones assist the refolding and degradation of misfolded proteins and prevention of the proteins’ aggregation. In the present study, we found that the expression of mutant γPKC-GFP increased the levels of heat-shock protein 70 (Hsp70) in SH-SY5Y cells. To elucidate the role of this elevation, we investigated the effect of siRNA-mediated knockdown of Hsp70 on the aggregation and cytotoxicity of mutant γPKC. Knockdown of Hsp70 exacerbated the aggregation and cytotoxicity of mutant γPKC-GFP by inhibiting this mutant’s degradation. These findings suggest that mutant γPKC increases the level of Hsp70, which protects cells from the mutant’s cytotoxicity by enhancing its degradation.     

Effect of Trehalose on the Properties of Mutant γPKC,Which Causes Spinocerebellar Ataxia Type 14, in Neuronal Cell Lines and Cultured Purkinje Cells

Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation, which induces apoptotic cell death. The disaccharide trehalose has been reported to inhibit aggregate formation and to alleviate symptoms in cellular and animal models of Huntington disease, Alzheimer disease, and prion disease. Here, we show that trehalose can be incorporated into SH-SY5Y cells and reduces the aggregation of mutant γPKC-GFP, thereby inhibiting apoptotic cell death in SH-SY5Y cells and primary cultured Purkinje cells (PCs). Trehalose acts by directly stabilizing the conformation of mutant γPKC without affecting protein turnover. Trehalose was also found to alleviate the improper development of dendrites in PCs expressing mutant γPKC-GFP without aggregates but not in PCs with aggregates. In PCs without aggregates, trehalose improves the mobility and translocation of mutant γPKC-GFP, probably by inhibiting oligomerization and thereby alleviating the improper development of dendrites. These results suggest that trehalose counteracts various cellular dysfunctions that are triggered by mutant γPKC in both neuronal cell lines and primary cultured PCs by inhibiting oligomerization and aggregation of mutant γPKC.     

In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis

In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.     

Biophysical characteristics of a new mutation on the KCNQ1 potassium channel (L251P) causing long QT syndrome

The congenital long QT syndrome (LQTS) is a hereditary cardiac disease characterized by prolonged ventricular repolarization, syncope, and sudden death. Mutations causing LQTS have been identified in various genes that encode for ionic channels or their regulatory subunits. Several of these mutations have been reported on the KCNQ1 gene encoding for a potassium channel or its regulatory subunit (KCNE1). In this study, we report the biophysical characteristics of a new mutation (L251P) in the transmembrane segment 5 (S5) of the KCNQ1 potassium channel. Potassium currents were recorded from CHO cells transfected with either wild type or mutant KCNQ1 in the presence or in the absence of its regulatory subunit (KCNE1), using the whole-cell configuration of the patch clamp technique. Wild-type KCNQ1 current amplitudes are increased particularly by KCNE1 co-expression but no current is observed with the KCNQ1 (L251P) mutant either in the presence or in the absence of KCNE1. Coexpressing KCNE1 with equal amount of cDNAs encoding wild type and mutant KCNQ1 results in an 11-fold reduction in the amplitude of potassium currents. The kinetics of activation and inactivation and the activation curve are minimally affected by this mutation. Our results suggest that the dominant negative effect of the P251L mutation on KCNQ1 channel explains the prolonged repolarization in patients carrying this mutation.     

Functional consequences of the G235R mutation in liver arginase leading to hyperargininemia

Hyperargininemia is a rare autosomal disorder that results from a deficiency in hepatic type I arginase. This deficiency is the consequence of random point mutations that occur throughout the gene. The G235R patient mutation has been proposed to affect the catalytic activity and structural integrity of the protein [D. E. Ash, L. R. Scolnick, Z. F. Kanyo, J. G. Vockley, S. D. Cederbaum, and D. W. Christianson (1998) Mol. Genet. Metab. 64, 243-249]. The G235R (patient) and G235A (control) arginase mutants of rat liver arginase have been generated to probe the effects of these point mutations on the structure and function of hepatic type I arginase. Both mutant arginases were trimeric by gel filtration, but the control G235A mutant had 56% of wild-type activity and the G235R mutant had less than 0.03% activity compared to the wild-type enzyme. The G235R mutant contained undetectable levels of tightly bound manganese as determined by electron paramagnetic resonance, while the G235A mutant had a Mn(II) stoichiometry of 2 Mn/subunit. Molecular modeling indicates that the introduction of an arginine residue at position 235 results in a major rearrangement of the metal ligands that compromise Mn(II) binding.     

Functional characterization of the P1059L mutation in the inositol 1,4,5-trisphosphate receptor type 1 identified in a Japanese SCA15 family

Spinocerebellar ataxia type 15 (SCA15) is a group of human neurodegenerative disorders characterized by a slowly progressing pure cerebellar ataxia. The inositol 1,4,5-trisphosphate (IP₃) receptor type 1 (IP₃R1) is an intracellular IP₃-induced Ca²⁺ release channel that was recently identified as a causative gene for SCA15. In most case studies, a heterozygous deletion of the IP₃R1 gene was identified. However, one Japanese SCA15 family was found to have a Pro to Leu (P1059L) substitution in IP₃R1. To investigate the effect of the P1059L mutation, we analyzed the channel properties of the mutant human IP₃R1 by expressing it in an IP₃R-deficient B lymphocyte cell line. The P1059L mutant was a functional Ca²⁺ release channel with a twofold higher IP₃ binding affinity compared to wild-type IP₃R1. The cooperative dependence of the Ca²⁺ release activity of the mutant on IP₃ concentration was reduced, but both wild-type and mutant receptors produced similar B cell receptor-induced Ca²⁺ signals. These results demonstrate that the Ca²⁺ release properties of IP₃R1 are largely unaffected by the P1059L mutation.     

Autosomal recessive retinitis pigmentosa and E150K mutation in the opsin gene

Retinitis pigmentosa (RP) is a heterogeneous group of hereditary disorders of the retina caused by mutation in genes of the photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Although there are over 100 identified mutations in the opsin gene associated with RP, only a few of them are inherited with the arRP pattern. E150K is the first reported missense mutation associated with arRP. This opsin mutation is located in the second cytoplasmic loop of this G protein-coupled receptor. E150K opsin expressed in HEK293 cells and reconstituted with 11-cis-retinal displayed an absorption spectrum similar to the wild type (WT) counterpart and activated G protein transducin slightly faster than WT receptor. However, the majority of E150K opsin showed a higher apparent molecular mass in SDS-PAGE and was resistant to endoglycosidase H deglycosidase. Instead of being transported to the plasma membrane, E150K opsin is partially colocalized with the cis/medial Golgi compartment markers such as GM130 and Vti1b but not with the trans-Golgi network. In contrast to the endoplasmic reticulum-retained adRP mutant, P23H opsin, Golgi-retained E150K opsin did not influence the proper transport of the WT opsin when coexpressed in HEK293 cells. This result is consistent with the recessive pattern of inheritance of this mutation. Thus, our study reveals a novel molecular mechanism for retinal degeneration that results from deficient export of opsin from the Golgi apparatus.     

Loss of Purkinje cells in the PKCγ H101Y transgenic mouse

Spinocerebellar ataxia type 14 (SCA14) is an autosomal, dominant neurodegenerative disorder caused by mutations in PKCγ. The objective of this study was to determine effects of PKCγ H101Y SCA14 mutation on Purkinje cells in the transgenic mouse. Results demonstrated that wild type PKCγ-like Purkinje cell localization of HA-tagged PKCγ H101Y mutant proteins, altered morphology and loss of Purkinje cells were observed in the PKCγ H101Y SCA14 transgenic mouse at four weeks of age. Failure of stereotypical clasping responses in the hind limbs of transgenic mice was also observed. Further, PKCγ H101Y SCA14 mutation caused lack of total cellular PKCγ enzyme activity, loss of connexin 57 phosphorylation on serines, and activation of caspase-12 in the PKCγ H101Y SCA14 transgenic mouse. Results clearly demonstrate a need for PKCγ control of gap junctions for maintenance of Purkinje cells. This is the first transgenic mouse to our knowledge which models a human SCA14 mutation.     

Structural and functional consequences of the mutation of a conserved arginine residue in alphaA and alphaB crystallins.

A point mutation of a highly conserved arginine residue in alphaA and alphaB crystallins was shown to cause autosomal dominant congenital cataract and desmin-related myopathy, respectively, in humans. To study the structural and functional consequences of this mutation, human alphaA and alphaB crystallin genes were cloned and the conserved arginine residue (Arg-116 in alphaA crystallin and Arg-120 in alphaB crystallin) mutated to Cys and Gly, respectively, by site-directed mutagenesis. The recombinant wild-type and mutant proteins were expressed in Escherichia coli and purified. The mutant and wild-type proteins were characterized by SDS-polyacrylamide gel electrophoresis, Western immunoblotting, gel permeation chromatography, fluorescence, and circular dichroism spectroscopy. Biophysical studies reveal significant differences between the wild-type and mutant proteins. The chaperone-like activity was studied by analyzing the ability of the recombinant proteins to prevent dithiothreitol-induced aggregation of insulin. The mutations R116C in alphaA crystallin and R120G in alphaB crystallin reduce the chaperone-like activity of these proteins significantly. Near UV circular dichroism and intrinsic fluorescence spectra indicate a change in tertiary structure of the mutants. Far UV circular dichroism spectra suggest altered packing of the secondary structural elements. Gel permeation chromatography reveals polydispersity for both of the mutant proteins. An appreciable increase in the molecular mass of the mutant alphaA crystallin is also observed. However, the change in oligomer size of the alphaB mutant is less significant. These results suggest that the conserved arginine of the alpha-crystallin domain of the small heat shock proteins is essential for their structural integrity and subsequent in vivo function.     

Hereditary pancreatitis caused by a novel PRSS1 mutation (Arg-122 --> Cys) that alters autoactivation and autodegradation of cationic trypsinogen.

Hereditary pancreatitis has been found to be associated with germline mutations in the cationic trypsinogen (PRSS1) gene. Here we report a family with hereditary pancreatitis that carries a novel PRSS1 mutation (R122C). This mutation cannot be diagnosed with the conventional screening method using AflIII restriction enzyme digest. We therefore propose a new assay based on restriction enzyme digest with BstUI, a technique that permits detection of the novel R122C mutation in addition to the most common R122H mutation, and even in the presence of a recently reported neutral polymorphism that prevents its detection by the AflIII method. Recombinantly expressed R122C mutant human trypsinogen was found to undergo greatly reduced autoactivation and cathepsin B-induced activation, which is most likely caused by misfolding or disulfide mismatches of the mutant zymogen. The K(m) of R122C trypsin was found to be unchanged, but its k(cat) was reduced to 37% of the wild type. After correction for enterokinase activatable activity, and specifically in the absence of calcium, the R122C mutant was more resistant to autolysis than the wild type and autoactivated more rapidly at pH 8. Molecular modeling of the R122C mutant trypsin predicted an unimpaired active site but an altered stability of the calcium binding loop. This previously unknown trypsinogen mutation is associated with hereditary pancreatitis, requires a novel diagnostic screening method, and, for the first time, raises the question whether a gain or a loss of trypsin function participates in the onset of pancreatitis.     

A mutation of keratin 18 within the coil 1A consensus motif causes widespread keratin aggregation but cell type-restricted lethality in mice

Mutations in genes encoding epidermal keratins cause skin disorders, while those in internal epithelial keratins, such as K8 and K18, are risk factors for liver diseases. The effect of dominant mutations in K8 or K18 during embryonic development and tissue homeostasis has not been examined so far. Here we demonstrate that the dominant mutation hK18 R89C, that is highly similar to hK14 R125C, causing EBS in humans, leads to cell type-specific lethality in mice, depending on the ratio of mutant to endogenous keratins. Mice expressing hK18 R89C in the absence of endogenous K19 and K18 died at mid-gestation from defects in trophoblast giant cells, accompanied by haematomas. A single, endogenous K18 allele rescued embryonic lethality but caused aggregation of keratins in all adult internal epithelia, surprisingly without spontaneous cell fragility. Closer analysis revealed that both filaments and aggregates coexisted in the same cell, depending on the ratio of mutant to endogenous keratins. Our results demonstrate that balanced overexpression of a wild-type keratin rescued the lethal consequences of a dominant-negative mutation. This has important implications for therapy approaches of keratinopathies, suggesting that suppressing the mutant allele is not necessary in vivo.     

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.     

A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling

Large scale sequencing of the human mu-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (beta-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, beta-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human mu-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo.     

Gly118Asp is a SCA14 founder mutation in the Dutch ataxia population

Missense mutations in the PRKCG gene have recently been identified in spinocerebellar ataxia 14 (SCA14) patients; these include the Gly118Asp mutation that we found in a large Dutch autosomal dominant cerebellar ataxia (ADCA) family. We subsequently screened the current Dutch ataxia cohort (approximately 900 individuals) for SCA14 mutations in the Cys2 region of the PRKCG gene. We identified the Gly118Asp mutation in another eight individuals from five small families. Haplotype analysis identified a shared chromosomal region surrounding the SCA14 gene, and genealogical research was able to link all these ADCA patients to a single common ancestor. We therefore confirmed that the Gly118Asp mutation is a SCA14 founder mutation in the Dutch ADCA population.     

The molecular basis of spinocerebellar ataxia type 48 caused by a de novo mutation in the ubiquitin ligase CHIP

The spinocerebellar ataxias (SCAs) are a class of incurable diseases characterized by degeneration of the cerebellum that results in movement disorder. Recently, a new heritable form of SCA, spinocerebellar ataxia type 48 (SCA48), was attributed to dominant mutations in STIP1 homology and U box-containing 1 (STUB1); however, little is known about how these mutations cause SCA48. STUB1 encodes for the protein C terminus of Hsc70 interacting protein (CHIP), an E3 ubiquitin ligase. CHIP is known to regulate proteostasis by recruiting chaperones via a N-terminal tetratricopeptide repeat domain and recruiting E2 ubiquitin-conjugating enzymes via a C-terminal U-box domain. These interactions allow CHIP to mediate the ubiquitination of chaperone-bound, misfolded proteins to promote their degradation via the proteasome. Here we have identified a novel, de novo mutation in STUB1 in a patient with SCA48 encoding for an A52G point mutation in the tetratricopeptide repeat domain of CHIP. Utilizing an array of biophysical, biochemical, and cellular assays, we demonstrate that the CHIP^A52G point mutant retains E3-ligase activity but has decreased affinity for chaperones. We further show that this mutant decreases cellular fitness in response to certain cellular stressors and induces neurodegeneration in a transgenic Caenorhabditis elegans model of SCA48. Together, our data identify the A52G mutant as a cause of SCA48 and provide molecular insight into how mutations in STUB1 cause SCA48.     

Identification and characterisation of a novel KCNQ1 mutation in a family with Romano-Ward syndrome

Romano-Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by approximately 92%, while IKs reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by approximately 60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native IKs currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.     

Structural distortion of p53 by the mutation R249S and its rescue by a designed peptide: implications for "mutant conformation"

Missense mutations in the DNA-binding core domain of the tumour suppressor protein p53 are frequent in cancer. Many of them result in loss of native structure. The mutation R249S is one of the six most common cancer-associated p53 mutations ("hot-spots"). As it is highly frequent in hepatocellular carcinoma, its rescue is an important therapeutic target. We have used NMR techniques to study the structural effects of the R249S mutation. The overall fold of the core domain is retained in R249S, and it does not take up a denatured "mutant conformation". However, the beta-sandwich had increased flexibility and, according to changes in chemical shift, there was local distortion throughout the DNA-binding interface. It is likely that the R249S mutation resulted in an ensemble of native and native-like conformations in a dynamic equilibrium. The peptide FL-CDB3 that was designed to rescue mutants of p53 by binding specifically to its native structure was found to revert the chemical shifts of R249S back towards the wild-type values and so reverse the structural effects of mutation. We discuss the implications for a rescue strategy and also for the analysis of antibody-binding data.