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PubMed1999

Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of beta-catenin, a component of the presenilin protein complex.

The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process.

Alzheimer Disease↗
PubMed1995

Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily.

Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.

3T3 Cells↗
PubMed1997

Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis.

It is not known whether mutations in the PKD1 gene cause autosomal dominant polycystic kidney disease (PKD) by an activating (gain-of-function) or an inactivating (loss-of-function) model. We analyzed DNA from cyst epithelial cells for loss of heterozygosity (LOH) in the PKD1 region of chromosome 16p13 using microsatellite markers. 29 cysts from four patients were studied. Five cysts from three patients had chromosome 16p13 LOH. Four of the cysts had loss of two chromosome 16p13 markers that flank the PKD1 gene. In two patients, microsatellite analysis of family members was consistent with loss of the wild-type copy of PKD1 in the cysts. In the third patient, 16p13 LOH was detected in three separate cysts, all of which showed loss of the same alleles. Chromosome 3p21 LOH was detected in one cyst. No LOH was detected in four other genomic regions. These results demonstrate that some renal cyst epithelial cells exhibit clonal chromosomal abnormalities with loss of the wild-type copy of PKD1. This supports a loss-of-function model for autosomal dominant PKD, with a germline mutation inactivating one copy of PKD1 and somatic mutation or deletion inactivating the remaining wild-type copy.

Chromosome Deletion↗
PubMed1999

Human nerve pathology caused by different mutational mechanisms of the PMP22 gene.

The study of the morphological phenotypes in patients with different mutations of the PMP22 gene gives additional insights into the role of the protein in myelin function. The pathology in young patients is in some aspects different from the pathology in older patients, providing essential and additional information about the early disease processes in humans induced by different PMP22 mutational mechanisms. Duplication of chromosome 17p11.2, causing an overexpression of the PMP22 gene, results in early hypermyelination. This suggests that PMP22 has a function in establishing the proper myelin thickness, probably in response to an axonal signal. Demyelination and remyelination with gradual formation of onion bulbs occur apparently as secondary processes, but these processes subside at the end of the first decade. PMP22 missense mutations usually result in a more severe phenotype showing an early hypomyelination and onion bulb formation, likely disturbing normal myelin formation and maintenance. The morphological differences with duplication cases underline the essential difference in pathogenesis between duplication and missense mutations despite the large overlap in clinical and electrophysiological phenotypes. The reciprocal deletion of chromosome 17p11.2, resulting in an underexpression of the PMP22 gene, is responsible for the clinical phenotype of HNPP. As the nerve abnormalities in HNPP show a marked resemblance to the changes in experimental and genuine human entrapment neuropathies, it is postulated that PMP22 has a function in adhesion of myelin lamellae, preventing mutual longitudinal sliding. Deletion of one PMP22 copy results in increased susceptibility for mechanical forces through which already minor nerve injuries might induce a longitudinal displacement of myelin lamellae. The frame shift mutation Gly94(insG) combines a loss of function like in the common deletion HNPP with a mild CMT1 phenotype, likely inducing a (mild) toxic gain of function by disturbing myelin formation and maintenance, comparable to the effect in missense mutations.

Animals↗
PubMed2001

p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fisher's exact test), more rarely mutated (P<0.006, Fisher's exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fisher's exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fisher's exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Amino Acid Sequence↗
PubMed2001

Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease.

Huntingtin is a 350-kilodalton protein of unknown function that is mutated in Huntington's disease (HD), a neurodegenerative disorder. The mutant protein is presumed to acquire a toxic gain of function that is detrimental to striatal neurons in the brain. However, loss of a beneficial activity of wild-type huntingtin may also cause the death of striatal neurons. Here we demonstrate that wild-type huntingtin up-regulates transcription of brain-derived neurotrophic factor (BDNF), a pro-survival factor produced by cortical neurons that is necessary for survival of striatal neurons in the brain. We show that this beneficial activity of huntingtin is lost when the protein becomes mutated, resulting in decreased production of cortical BDNF. This leads to insufficient neurotrophic support for striatal neurons, which then die. Restoring wild-type huntingtin activity and increasing BDNF production may be therapeutic approaches for treating HD.

3T3 Cells↗
PubMedDate not supplied

Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining.

The DNA-dependent protein kinase functions in the repair of DNA double strand breaks (DSBs) and in V(D)J recombination. To gain insight into the function of DNA-PK in this process we have carried out a mutation analysis of Ku80 and DNA-PKcs. Mutations at multiple sites within the N-terminal two thirds of Ku80 result in loss of Ku70/80 interaction, loss of DNA end-binding activity and inability to complement Ku80 defective cell lines. In contrast, mutations in the carboxy terminal region of the protein do not impair DNA end-binding activity but decrease the ability of Ku to activate DNA-PK. To gain insight into important functional domains within DNA-PKcs, we have analysed defective mutants, including the mouse scid cell line, and the rodent mutants, irs-20 and V-3. Mutational changes in the carboxy terminal region have been identified in all cases. Our results strongly suggest that the C-terminus of DNA-PKcs is required for kinase activity.

Animals↗
PubMed1999

[Growing genes cause neurological diseases].

A growing number of hereditary neurodegenerative disorders have been found to be caused by expansion of trinucleotide repeats. A smaller number of diseases such as fragile X syndrome, myotonic dystrophy, and Friedreich's ataxia, have been found to be due to expansions in non-coding DNA. In a large group of diseases, the expansion consists of CAG repeats in the coding region of the gene, producing an expanded polyglutamine sequence in the protein. Nine diseases have so far been identified as belonging to this group: Huntington's disease, spinobulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA), autosomal dominant "pure" spastic paraplegia (ADPSP), and five forms of spinocerebellar ataxia (SCA 1,2,3,6 and 7). Except for SBMA, all of the CAG repeat disorders are characterised by autosomal dominant heredity and anticipation (i.e., earlier onset age and increasing severity in successive generations). The mutated protein causes disease via an as yet unidentified gain-of-function mechanism in specific subsets of neurones. Today, DNA analysis permits the diagnosis of a trinucleotide disease in individual cases.

Anticipation, Genetic↗
PubMed1997

Superoxide-dependent peroxidase activity of H48Q: a superoxide dismutase variant associated with familial amyotrophic lateral sclerosis.

Approximately 20% of cases of familial amyotrophic lateral sclerosis are caused by dominant mutations in the Cu,Zn superoxide dismutase. One such mutant, in which histidine #48 has been replaced by glutamine (H48Q), exhibits a novel activity. It can react sequentially with O2- and H2O2 to generate a potent oxidant at its active site, possibly Cu(II)-OH, which then can oxidize urate to the corresponding radical. This O2- -dependent peroxidase activity exerted on a substrate peculiar to motor neurons may be the toxic gain of function which leads to the deleterious consequences of this mutation. G93A, G93R, and E100G were also examined and found not to exert this O2- -dependent peroxidase activity.

Amyotrophic Lateral Sclerosis↗
PubMed1999

A novel follicle-cell-dependent dominant female sterile allele, StarKojak, alters receptor tyrosine kinase signaling in Drosophila.

We describe a new dominant allele, StarKojak, that alters receptor tyrosine kinase signaling in the follicle cells and in the eyes in Drosophila. We isolated StarKojak in a screen for follicle-cell-dependent dominant female sterile mutations. We show that StarKojak and revertants of StarKojak do not complement Star loss-of-function mutations. We propose that StarKojak is a novel type of allele of Star that has both dominant gain-of-function phenotypes early in development and dominant loss-of-function phenotypes later in development. Star encodes a putative transmembrane protein that has previously been shown to be a critical component of the epidermal growth factor receptor tyrosine kinase signaling pathway. Early in oogenesis, Star mRNA expression is higher in StarKojak egg chambers than in wild-type egg chambers, consistent with its gain-of-function phenotype. Later in oogenesis, Star mRNA expression is lower in StarKojak follicle cells than in wild-type follicle cells, consistent with its loss-of-function phenotype. By genetically analyzing StarKojak and its revertants, we present evidence that Star is involved in anterior-posterior axis formation both in the female germline cells and in the somatic follicle cells. We also demonstrate that at least part of the dominant female sterile phenotype of StarKojak is restricted to the posterior-pole follicle cells. We propose that Star functions by processing pro-Gurken to mature Gurken, which is thereby released in the region between the oocyte and the follicle cells and binds to the epidermal growth factor receptor in the follicle cells.

Alleles↗
PubMed2002

Gastrointestinal stromal tumour of the oesophagus: significance of immunohistochemical and genetic analyses of the c-kit gene.

Oesophageal gastrointestinal stromal tumours (GISTs) are rare in comparison to those of the stomach and intestines. Recently, it has been clarified that mutations of the c-kit gene resulting in gain of function might be associated with histogenesis of this type of tumour arising in the stomach and intestines. We describe an oesophageal GIST on immunohistochemical and genetic analyses of the gene. A 71-year-old man had an intramural tumour of the middle third of the oesophagus. Tumour cells were composed predominantly of spindle-shaped and partially epithelioid cells. They were diffusely positive for CD117. Six base deletion resulting in in-frame mutation of the c-kit gene was confirmed at codon 556-558 (cag tgg aag to cag) of exon 11. Patients with mutations of the c-kit gene revealed worse prognoses in GISTs arising from other locations. A long-term follow-up observation is needed for the case.

Aged↗
PubMed1995

Activation of Bruton's tyrosine kinase (BTK) by a point mutation in its pleckstrin homology (PH) domain.

Bruton's tyrosine kinase (BTK) is a nonreceptor tyrosine kinase critical for B cell development and function. Mutations in BTK result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Using a random mutagenesis scheme, we isolated a gain-of-function mutant called BTK* whose expression drives growth of NIH 3T3 cells in soft agar. BTK* results from a single point mutation in the pleckstrin homology (PH) domain, where a Glu is replaced by Lys at residue 41. BTK* shows an increase in phosphorylation on tyrosine residues and an increase in membrane targeting. Transforming activity requires kinase activity, a putative autophosphorylation site, and a functional PH domain. Mutation of the SH2 or SH3 domains did not affect the activity of BTK*. Expression of BTK* could also relieve IL-5 dependence of a B lineage cell line. These results show that transformation activation and regulation of BTK are critically dependent on the PH domain.

3T3 Cells↗
PubMed2004

Dominant, gain-of-function mutant produced by truncation of RPGR.

PURPOSE: The retinitis pigmentosa GTPase regulator (RPGR) is essential in the maintenance of photoreceptor viability. Mutations in the X-linked RPGR gene have generally been assumed to be recessive. This study was undertaken to investigate whether certain mutant RPGR alleles may act dominantly. METHODS: An RPGR transgene representing the RPGR ORF15 variant was placed under a non-tissue-specific promoter and introduced into transgenic mice. The transgene was crossed into both a wild type (WT) and an RPGR null background. Its expression was analyzed by RT-PCR, immunoblot analysis, and immunofluorescence. Photoreceptor survival was assessed by electroretinography and histology. RESULTS: The RPGR transgene transcript underwent photoreceptor-specific, alternative splicing involving the purine-rich region of the ORF15 exon, generating a shortened mRNA and a premature stop codon. This truncation mutant caused more rapid photoreceptor degeneration than that in the RPGR null (knockout) mutant. The disease course was similar, whether the transgene was coexpressed with WT RPGR or expressed alone in the RPGR null background. CONCLUSIONS: Certain truncated forms of RPGR can behave as a dominant, gain-of-function mutant. These data suggest that human RPGR mutations are not necessarily null and some may also act as dominant alleles, leading to a more severe phenotype than a null mutant.

Alternative Splicing↗
PubMed1997

Phosphorylation of the G protein alpha-subunit, G alpha 2, of Dictyostelium discoideum requires a functional and activated G alpha 2.

The G alpha 2-subunit of Dictyostelium discoideum is essential to the initial stage of the cell's developmental life cycle. In response to the extracellular chemoattractant, cAMP, G alpha 2 is activated and transiently phosphorylated on serine-113 [Chen et al. (1994): J Biol Chem 269:20925-20930]. The role of G alpha 2 phosphorylation remains elusive; cells expressing the S113A, nonphosphorylated mutation of G alpha 2 appear to proceed through the developmental phase normally. To gain insight into the function of G alpha 2 phosphorylation, the conditions for G alpha 2 phosphorylation were examined using a variety of alpha-subunit point mutations and chimeras. Mutations that block the G protein activation cycle prior to or at the hydrolysis of GTP (G alpha 2-S45A, G alpha 2-G207A, and G alpha 2-Q208L) preclude G alpha 2 phosphorylation in vivo. Phosphorylation of the G alpha 2-Q208L mutation does however occur in an in vitro phosphorylation assay. It appears that G alpha 2 phosphorylation, shown previously in vivo to require the cAMP receptor, also requires signaling through the G2 pathway. Results from the in vitro assay suggest that the substrate for phosphorylation is the alpha-subunit monomer.

Animals↗
PubMedDate not supplied

Use of gain-of-function study to delineate the roles of crumbs in Drosophila eye development.

The cell polarity gene, crumbs (crb)), has been shown to participate in the development and degeneration of the Drosophila retina. Mutations in CRB1, the human homologue of Drosophila crb, also result in retinitis pigmentosa and Leber congenital amaurosis. In this study, we used the gain-of-function approach to delineate the roles of CRB in developing Drosophila eye. In the third-instar larval stage, eye development is initiated with photoreceptor differentiation and positioning of photoreceptor nuclei in the apical cellular compartment of retinal epithelium. In the pupal stage, differentiated photoreceptors begin to form the photosensitive structures, the rhabdomeres, at their apical surface. Using GMR-Gal4 to drive overexpression of the Crb protein at the third-instar eye disc, we found that differentiation of photoreceptors was disrupted and the nuclei of differentiated photoreceptors failed to occupy the apical compartment. Using HS-Gal4 to drive Crb overexpression in pupal eyes resulted in interference with extension of the adherens junctions and construction of the rhabdomeres, and these defects were stage-dependent. This gain-of-function study has enabled us to delineate the roles of Crb at selective stages of eye development in Drosophila.

Adherens Junctions↗
PubMed2003

NTE: one target protein for different toxic syndromes with distinct mechanisms?

Epidemics of organophosphate-induced delayed neuropathy (OPIDN) have paralysed thousands of people. This syndrome of nerve axon degeneration is initiated by organophosphates which react with neuropathy target esterase (NTE). Dosing experiments with adult chickens raise the possibility that OPIDN is initiated by a gain-of-function mechanism. By contrast, loss of NTE function by mutation causes massive apoptosis in Drosophila brain. Now, Winrow et al. show that nte(-/-) mice die by mid-gestation, but nte(+/-) mice appear hyperactive and are more sensitive than wild-type mice to a fatal form of OP toxicity. Thus, different toxic syndromes may be initiated via a single target protein.

Acetylcholinesterase↗
PubMed2006

One hundred years of high-throughput Drosophila research.

From the beginning, Drosophila was a high-throughput model organism. Unbiased and genome-wide efforts ranging from Morgan's search for spontaneous mutations and subsequent saturating loss-of-function and gain-of-function screens up to more recent techniques such as microarrays, proteomics and cellular assays have been and will continue to be the backbone of Drosophila research. Integrating these large datasets is one of the next challenges. However, once achieved, a plethora of information far exceeding the information content of the singular experiments will be revealed. Several high-throughput techniques and experimental strategies highlighting the unbiased and integrative nature of Drosophila research during the last century will be discussed.

Animals↗
PubMed2006

Oculopharyngeal muscular dystrophy: potential therapies for an aggregate-associated disorder.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, autosomal dominant disease caused by the abnormal expansion of a polyalanine tract within the coding region of poly(A) binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. The mechanism by which the polyalanine expansion mutation in PABN1 causes disease is unclear. However, the mutation is thought to confer a toxic gain-of-function on the protein. Despite controversy over the role of aggregates, it has been consistently shown that agents that reduce aggregate load in cell models of OPMD also reduce levels of cell death. Recently generated animal models of OPMD will help elucidate the mechanism of disease and allow the trial of potential therapeutics. Indeed, administration of known anti-aggregation drugs attenuated muscle weakness in an OPMD mouse model. This suggests that anti-aggregation therapies may be beneficial in OPMD.

Animals↗