Recombinant Mutant Proteins Research Articles

A Novel Breast Cancer–Associated BRIP1 (FANCJ/BACH1) Germ-line Mutation Impairs Protein Stability and Function

BRCA1-interacting protein 1 (BRIP1; FANCJ/BACH1), which encodes a DNA helicase that interacts with BRCA1, has been suggested to be a low-penetrance breast cancer predisposing gene. We aimed to assess whether BRIP1 mutations contribute to breast cancer susceptibility in our population and, if so, to investigate the effect of such mutation(s) on BRIP1 function. A series of 49 breast/ovarian cancer families, devoid of a BRCA1/BRCA2 mutation, were screened for BRIP1 mutations. Functional analyses, including coimmunoprecipitation and stability assays, were employed to further characterize a previously unreported variant. Five sequence alterations were identified, of which four had been already described. Herein, we report a novel BRIP1 germ-line mutation identified in a woman with early-onset breast cancer. The mutation consists of a 4-nucleotide deletion (c.2992-2995delAAGA) in BRIP1 exon 20 that causes a shift in the reading frame, disrupts the BRCA1-binding domain of BRIP1, and creates a premature stop codon. Functional analysis of the recombinant mutant protein in transfected cells showed that the truncation interferes with the stability of the protein and with its ability to interact with BRCA1. Loss of the wild-type BRIP1 allele with retention of the mutated one was observed in the patient's breast tumor tissue. These results, by showing that the newly identified BRIP1 c.2992-2995delAAGA mutation is associated with instability and functional impairment of the encoded protein, provide further evidence of a breast cancer-related role for BRIP1.

Characterization of Dengue Virus Complex-Specific Neutralizing Epitopes on Envelope Protein Domain III of Dengue 2 Virus

The surface of the mature dengue virus (DENV) particle is covered with 180 envelope (E) proteins arranged as homodimers that lie relatively flat on the virion surface. Each monomer consists of three domains (ED1, ED2, and ED3), of which ED3 contains the critical neutralization determinant(s). In this study, a large panel of DENV-2 recombinant ED3 mutant proteins was used to physically and biologically map the epitopes of five DENV complex-specific monoclonal antibodies (MAbs). All five MAbs recognized a single antigenic site that includes residues K310, I312, P332, L389, and W391. The DENV complex antigenic site was located on an upper lateral surface of ED3 that was distinct but overlapped with a previously described DENV-2 type-specific antigenic site on ED3. The DENV complex-specific MAbs required significantly higher occupancy levels of available ED3 binding sites on the virion, compared to DENV-2 type-specific MAbs, in order to neutralize virus infectivity. Additionally, there was a great deal of variability in the neutralization efficacy of the DENV complex-specific MAbs with representative strains of the four DENVs. Overall, the differences in physical binding and potency of neutralization observed between DENV complex- and type-specific MAbs in this study demonstrate the critical role of the DENV type-specific antibodies in the neutralization of virus infectivity.

Characterization of Molecular Mechanisms Underlying Mutations in Dystrophic Epidermolysis Bullosa Using Site-directed Mutagenesis

Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 alpha chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients.

A novel mutation in AlphaA-crystallin (CRYAA) caused autosomal dominant congenital cataract in a large Chinese family

Hereditary cataract is a phenotypically and genetically heterogeneous lens disease that is responsible for a significant proportion of the visual impairment and blindness that occurs in children. In a five-generation Chinese family with autosomal dominant inherited congenital cataract, clinical examination showed three cataract phenotypes: punctuate, nuclear, and total cataracts. Linkage analysis was performed and positive two-point LOD scores (with maximum of 4.43 and 4.27 at theta=0) were obtained for markers D21S1411 and D21S1890 on chromosome 21q22.3, flanking the CRYAA (alphaA-crystallin-encoding gene) locus. Sequencing of CRYAA revealed a novel heterozygous G>A transition (c.346G>A) in exon 3 that cosegregated with the disease phenotype and results in a conservative substitution of Arg to His at codon 116 (p.R116H). To understand the molecular basis of cataract formation, mutant and wild-type alphaA-crystallins were expressed in E. coli. RP-HPLC (reverse phase-high-performance liquid chromatography) suggested an increased hydrophobicity of the mutant recombinant protein, compared to that of wild-type alphaA-crystallins. Furthermore, loss of chaperone activity of the mutant was seen in DTT (DL-dithiothreitol)-induced insulin aggregation assay. FPLC (fast protein liquid chromatography) purification showed that the His-116 mutant protein had increased binding affinity to lysozyme. Gain of activated lysozyme binding, elevation of hydrophobicity and loss of chaperone activity of the mutant protein may be some of the molecular mechanisms underlying cataract in this large family.

Immunization with an engineered mutant trans-sialidase highly protects mice from experimental Trypanosoma cruzi infection: A vaccine candidate

Chagas' disease is a major tropical disease for which a cure for chronic phase does not exist yet. Trypanosoma cruzi trans-sialidase (TS) seems to be involved in relevant processes such as infectivity, host survival and, very importantly, disease pathogenesis. In this study, we show that mice vaccinated with an engineered enzymatically deficient mutant TS containing the catalytic domain without the immunodominant SAPA (Shed Acute Phase Antigen) repeats, were highly protected against T. cruzi infection. Adult male BALB/c mice were immunized with mutant protein, purified from Pichia pastoris yeast, using three inoculations in Freund's adjuvant. All immunized mice were protected against challenge with a lethal dose of T. cruzi trypomastigotes. The protected immunized mice developed no clinical or tissue evidence of infection throughout the study. In contrast, 60-90% mortality and 100% occurrence of myocardial lesions were observed in the non-immunized counterparts. Titers of circulating antibody against TS did not correlate with protection, while anti-SAPA antibodies were coincident with disease severity. Further studies indicated that a single inoculation of mutant recombinant protein in Freund's complete adjuvant was not associated with blood or organic alterations, per se. Mutant TS vaccination seems to be a promising tool for immune intervention strategies in Chagas' disease, aimed at preventing T. cruzi-related heart tissue damage.

Food Biochemical Study on Fructans and Related Synthesis Enzymes

There are more than 500 kinds of oligosaccharide occurring in nature, and all of these are synthesized by chemical and enzymatic reactions. Various studies have recently identified physiological and physical applications of such oligosaccharides in agricultural chemistry, nutrition and medicine. New technologies for the production of oligosaccharides from natural resources have been developed in the food industry and several oligosaccharides are now produced on a large scale as ingredients in animal feed. We have investigated the isolation of fructo-oligosaccharides, as well as the enzymatic synthesis of oligosaccharides that have functional activities as tertiary functional ingredients in food. We have studied the purification and characterization of several fructosyltransferases from asparagus roots and onion bulbs, including sucrose: sucrose 1-fructosyltransferase (1-SST), fructan: fructan 1-fructosyltransferase (1-FFT), and the novel enzyme fructan:fructan 6G-fructosyltransferase (6G-FFT). We have also reported that asparagus 1-FFT synthesized new functional oligosaccharides elongated with one or two additional fructose units by fructosyltransfer from 1-kestose to 4G-β-D-galactosylsucrose, and this compound selectively stimulates the growth of Bifidobacteria. For industrial applications, we attempted the isolation and expression of cDNAs encoding 6G-FFT, 1-FFT and 1-SST from asparagus plants. The cDNAs encoding 6G-FFT, 1-FFT and 1-SST were isolated from a cDNA library of asparagus leaves or roots, and the isolated cDNAs were designated aoft1, aoft2 and aoft3, respectively. The deduced amino acid sequences of these cDNAs showed high homology with those of plant fructosyltransferases. Expression of these cDNAs was performed using Pichia pastoris. The recombinant protein from Pichia transformed with aoft1 produced 1F,6G-di-β-D-fructofuranosylsucrose, neokestose and sucrose from 1-kestose, while the transformant with an empty vector produced no saccharides. These results confirmed that 6G-FFT was expressed in P. pastoris. Similarly, the recombinant protein from Pichia transformed with aoft2 produced nystose from 1-kestose and the recombinant protein with aoft3 produced 1-kestose from sucrose. These results confirmed that 1-FFT and 1-SST were expressed in P. pastoris, and that the recombinant proteins had enzymatic properties similar to those of 6G-FFT, 1-FFT and 1-SST from asparagus roots. We then examined the conversion of substrate specificity from 6G-FFT to 1-FFT by point mutations in the β-fructosidase motif. The asparagine in this motif in aoft1 was changed to serine, and the mutant recombinant protein was characterized. We found that this amino acid substitution in wild-type aoft1 changed the substrate specificity from 6G-FFT to 1-FFT. Finally, we studied the activities of the main enzymes involved in the synthesis and hydrolysis of fructo-oligosaccharides during the post-harvest life of onion bulbs and asparagus spears, and discuss the mechanisms triggering these enzyme activities, as well as the mechanisms by which fructo-oligosaccharides contribute to the quality and perishability of the vegetables.

Single Residue Determines the Specificity of Neutrophil Elastase for Shigella Virulence Factors

Human neutrophil elastase (NE) is a key host defense protease that cleaves virulence factors of Gram-negative bacteria. NE and cathepsin G (CG) are chymotrypsin-like serine proteases with sequence and structural similarities, and both are abundant in neutrophil granules. Unlike NE, CG does not cleave virulence factors of enteric bacteria. Through structure–function analysis, we identified regions in NE that are essential for cleaving Shigella virulence proteins. NE residues at eight different positions were replaced with analogous amino acids in CG or with alanine. Functional analysis of recombinant mutant proteins showed that a single residue at position 98 and multiple amino acid stretches in the three different regions 58A–61, 163–181, and 216–224 determine NE specificity. These NE mutants cleaved the CG-specific, but not the NE-specific, synthetic peptide substrate and did not degrade Shigella virulence factors. Interestingly, exchanging the amino acid at position 98 in CG for the NE equivalent enabled this CG mutant to cleave Shigella virulence factors. Analysis of the NE proteolytic products of the Shigella virulence factor IpaB shows that NE has specific cleavage sites. These results indicate that Shigella virulence factor specificity maps to a distinct region close to NE's active site.

The Severity of Bleeding Tendency in 5 Patients with Factor V Deficiency Due to Distinct Kinds of Factor V Mutations.

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. It is poor correlation between FV levels in plasma and the severity of bleeding tendency. In the present study, we identified 5 mutations in the FV gene (F5) in 5 unrelated Japanese patients with reduced plasma FV activities associated with inherited FV deficiency. Their bleeding tendencies varied in severity from asymptomatic to severe. We hypothesized that the severity of bleeding symptoms with severe FV deficiency is correlated with FV levels in platelets, and performed recombinant mutant proteins expression experiments and analyzed of platelet FV. The data concerning 5 patient's FV levels in plasma and the bleeding symptoms are given in Table. The F5 mutations in 5 Japanese patients were identified by direct sequencing. One of the 5 patients was a compound heterozygote for FV mutations and carried a 5-base pair (bp) deletion in exon 22 (del.ACCCT) and V1813M. The other 4 patients were homozygous for a D68H, N468S, V1813M, and R2174L mutations, respectively. Four mutations except V1813M are newly identified mutations. These mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA, and the wild-type and mutant FV proteins were expressed in HEK293 cells. In the conditioned media, FV specific activities of the FV-D68H, FV-N468S, FV-V1813M, FV-R2174L, and 5bp del. mutants were an approximately 22%, 81%, 28%, 40%, and 19% of wild-type, respectively. On the other hand, analysis of platelet from patient by using RT-PCR showed that platelet F5 mRNA of FV-R2174L and FV-N468S was equal amount to that of normal subjects, although the amount of FV-V1813M platelet F5 mRNA was reduced. Platelet FV protein from patients was analyzed by western blotting and ELISA. Although the amount of platelet FV-R2174L protein was equal to that of normal platelets, platelet FV-V1813M protein was considerably reduced. In addition, the amount of FV-N468S protein in platelet was observed between that of normal subjects and FV-V1813M. These results indicate that the fact that sufficient amounts of FV are stored in platelet is required for local hemostasis. The results of FV-R2174L suggest that both functionality and amount of FV-R2174L in platelet is enough to cope with local bleeding, resulting very mild bleeding tendency. On the basis of present findings, we conclude that the severity of bleeding due to severe FV deficiency is correlated with not only plasma FV level, but also platelet FV.Five patient's FV levels in plasma and the bleeding symptomsPatient No.MutationFV activity (%)FV antigen (%)Bleeding symptoms1D68H44asymptomatic2N468S33asymptomatic3V1813M & 5-bp deletion<19severe4V1813M<14moderate5R2174L15very mild

Structural and Functional Characterization of Recombinant Matrilin-3 A-domain and Implications for Human Genetic Bone Diseases

Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the beta-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn(2+). In the presence of Zn(2+) the A-domain adopts a more stable "tighter" conformation. However, after the removal of Zn(2+) a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more "relaxed" conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein. We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn(2+)-dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen.

Dimerization and Actin-bundling Properties of Villin and Its Role in the Assembly of Epithelial Cell Brush Borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

Specific Protein Domains Mediate Cooperative Assembly of HuR Oligomers on AU-rich mRNA-destabilizing Sequences

The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR.ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR.ARE complexes in solution.

A novel method for analyzing phosphoproteins using SELDI‐TOF MS in combination with a series of recombinant proteins

A novel SELDI-TOF MS-based method for analyzing phosphoproteins was developed using a series of recombinant wild-type and mutant ribosomal P2 proteins. We demonstrated that the phosphorylation status of the overexpressed proteins in cells was easily and rapidly confirmed using this method. The ribosomal P2 protein contained two phosphorylation sites, which were sequentially phosphorylated in vivo. We also quantitatively detected the phosphoprotein by using SELDI-TOF MS.

Different Amino-Acid Substitutions Confer Insecticide Resistance Through Acetylcholinesterase 1 Insensitivity in &lt;I&gt;Culex vishnui&lt;/I&gt; and &lt;I&gt;Culex tritaeniorhynchus&lt;/I&gt; (Diptera: Culicidae) from China

Insecticide resistance owing to insensitive acetylcholinesterase (AChE)1 has been reported in several mosquito species, and only two mutations in the ace-1 gene have been implicated in resistance: 119S and 331W substitutions. We analyzed the AChE1 resistance status of Culex vishnui (Theobald) and Culex tritaeniorhynchus Giles sampled in various regions of China. These two species displayed distinct mutations leading to AChE1 insensitivity; the 119S substitution in resistant C. vishnui mosquitoes and the 331W substitution in resistant C. tritaeniorhynchus. A biochemical test was validated to detect the 331W mutation in field samples. The comparison of the recombinant G119S and 331W mutant proteins produced in vitro with the AChE1 extracted from resistant mosquitoes indicated that the AChE1 insensitivity observed could be specifically attributed to these substitutions. Comparison of their biochemical characteristics indicated that the resistance conferred by these mutations depends on the insecticide used, regardless of its class. This resistance seemed to be fixed in the Cx. tritaeniorhynchus populations sampled in a 2000-km transect, suggesting a very high level of insecticide application or a low fitness cost associated with this 331W mutation.

Clearance mechanisms of von Willebrand factor and factor VIII.

Clearance mechanisms of von Willebrand factor and factor VIII.

A Substrate Specificity-determining Unit of Three Lin12-Notch Repeat Modules Is Formed in Trans within the Pappalysin-1 Dimer and Requires a Sequence Stretch C-terminal to the Third Module

Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP-A cleaves IGFBP-4 and IGFBP-5, whereas PAPP-A2 cleaves only IGFBP-5. The pappalysins contain three Lin12-Notch repeat (LNR1-3) modules, previously considered unique to the Notch receptor family in which they function to regulate receptor cleavage. In contrast to the Notch receptor where three LNR modules are tandemly arranged, LNR3 is separated by more than 1000 residues from LNR1-2 in the pappalysin sequence. Each of the three LNR modules of PAPP-A is required for proteolysis of IGFBP-4, but not IGFBP-5. However, we here find that a C-terminal truncated variant of PAPP-A, which lacks LNR3 and therefore activity against IGFBP-4, cleaves IGFBP-4 when co-expressed with a PAPP-A variant, which is mutated in the active site. This suggests that LNR3 from the inactive subunit interacts in trans with LNR1-2 of the truncated PAPP-A subunit to form a functional trimeric LNR unit. We also show that formation of such a functional LNR unit depends on dimerization, as dissociation of a mutated non-covalent PAPP-A dimer results in reduced activity against IGFBP-4, but not IGFBP-5. Using PAPP-A/PAPP-A2 chimeras, we demonstrate that PAPP-A2 LNR1-2, but not LNR3, are functionally conserved with respect to IGFBP proteolysis. Additionally, we find that a sequence stretch C-terminal to LNR3 and single residues (Asp1521, Arg1529, and Asp1530) within this are required for LNR functionality.

Focal Adhesion Kinase Is Negatively Regulated by Phosphorylation at Tyrosine 407

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.

Enzyme enhancement activity of N-octyl-β-valienamine on β-glucosidase mutants associated with Gaucher disease

Gaucher disease (GD), caused by a defect of β-glucosidase (β-Glu), is the most common form of sphingolipidosis. We have previously shown that a carbohydrate mimic N-octyl-β-valienamine (NOV), an inhibitor of β-Glu, could increase the protein level and enzyme activity of F213I mutant β-Glu in cultured GD fibroblasts, suggesting that NOV acted as a pharmacological chaperone to accelerate transport and maturation of this mutant enzyme. In the current study, NOV effects were evaluated in GD fibroblasts with various β-Glu mutations and in COS cells transiently expressing recombinant mutant proteins. In addition to F213I, NOV was effective on N188S, G202R and N370S mutant forms of β-Glu, whereas it was ineffective on G193W, D409H and L444P mutants. When expressed in COS cells, the mutant proteins as well as the wild-type protein were localized predominantly in the endoplasmic reticulum and were sensitive to Endo-H treatment. NOV did not alter this localization or Endo-H sensitivity, suggesting that it acted in the endoplasmic reticulum. Profiling of N-alkyl-β-valienamines with various lengths of the acyl chain showed that N-dodecyl-β-valienamine was as effective as NOV. These results suggest a potential therapeutic value of NOV and related compounds for GD with a broad range of β-Glu mutations.

Therapeutic effects of immunization with mutant superoxide dismutase in mice models of amyotrophic lateral sclerosis

There is emerging evidence for the existence of secretory pathways for superoxide dismutase (SOD1) mutants linked to amyotrophic lateral sclerosis (ALS) and for neurotoxicity of extracellular mutant SOD1. This evidence led us to test immunization protocols aiming to reduce the burden of extracellular SOD1 mutants in nervous tissue of mice models of ALS, by using bacterially purified recombinant SOD1 mutant protein as an immunogen. First, a vaccination was tested on a G37R SOD1 mouse strain with late-onset disease exhibiting levels of mutant SOD1 protein at 4-fold higher than normal SOD1 levels. Repeated injections of adjuvant/SOD1 mutant with a final booster injection before symptoms at 6 months of age were effective in delaying disease onset and extending the life span of G37R SOD1 mice by >4 weeks. Western blot analysis with a monoclonal antibody specific to mutant SOD1 forms provided evidence of clearance of SOD1 species in the spinal cord of vaccinated G37R SOD1 mice. In contrast, this vaccination approach failed to confer significant protection in G93A SOD1 mice with extreme overexpression of mutant SOD1. Nonetheless, a passive immunization through intraventricular infusion of purified anti-human SOD1 antibody with osmotic minipump succeeded in alleviating disease symptoms and prolonging the life span of G93A SOD1 mice. From these results, we propose that immunization strategies should be considered as potential avenues for treatment of familial ALS caused by SOD1 mutations.

A critical β6–β7 loop in the pleckstrin homology domain of ceramide kinase

CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.

Sulfonylureas Correct Trafficking Defects of Disease-causing ATP-sensitive Potassium Channels by Binding to the Channel Complex

ATP-sensitive potassium (K(ATP)) channels mediate glucose-induced insulin secretion by coupling metabolic signals to beta-cell membrane potential and the secretory machinery. Reduced K(ATP) channel expression caused by mutations in the channel proteins: sulfonylurea receptor 1 (SUR1) and Kir6.2, results in loss of channel function as seen in congenital hyperinsulinism. Previously, we reported that sulfonylureas, oral hypoglycemic drugs widely used to treat type II diabetes, correct the endoplasmic reticulum to the plasma membrane trafficking defect caused by two SUR1 mutations, A116P and V187D. In this study, we investigated the mechanism by which sulfonylureas rescue these mutants. We found that glinides, another class of SUR-binding hypoglycemic drugs, also markedly increased surface expression of the trafficking mutants. Attenuating or abolishing the ability of mutant SUR1 to bind sulfonylureas or glinides by the following mutations: Y230A, S1238Y, or both, accordingly diminished the rescuing effects of the drugs. Interestingly, rescue of the trafficking defects requires mutant SUR1 to be co-expressed with Kir6.2, suggesting that the channel complex, rather than SUR1 alone, is the drug target. Observations that sulfonylureas also reverse trafficking defects caused by neonatal diabetes-associated Kir6.2 mutations in a way that is dependent on intact sulfonylurea binding sites in SUR1 further support this notion. Our results provide insight into the mechanistic and structural basis on which sulfonylureas rescue K(ATP) channel surface expression defects caused by channel mutations.