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Results for “Gain of Function Mutation”

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At least 1,117 records · Page 62Linked to original sources
PubMed2003

An enhanced U6 promoter for synthesis of short hairpin RNA.

Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo and, therefore, may be developed to treat diseases caused by dominant, gain-of-function type of gene mutations. These diseases develop in people bearing one mutant and one wild-type gene allele. While the mutant is toxic, the wild-type performs important functions. Thus, the ideal therapy must selectively silence the mutant but maintain the wild-type expression. To achieve this goal, we designed an shRNA that selectively silenced a mutant Cu,Zn superoxide dismutase (SOD1(G93A)) allele that causes amyotrophic lateral sclerosis. However, the efficacy of this shRNA was relatively modest. Since the allele-specific shRNA has to target the mutation site, we could not scan other regions of SOD1 mRNA to find the best silencer. To overcome this problem, we sought to increase the dose of this shRNA by enhancing the Pol III promoter. Here we demonstrate that the enhancer from the cytomegalovirus immediate-early promoter can enhance the U6 promoter activity, the synthesis of shRNA and the efficacy of RNA interference (RNAi). Thus, this enhanced U6 promoter is useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region.

Base Sequence↗
PubMed2004

Dosage-sensitive requirement for mouse Dll4 in artery development.

Involvement of the Notch signaling pathway in vascular development has been demonstrated by both gain- and loss-of-function mutations in humans, mice, and zebrafish. In zebrafish, Notch signaling is required for arterial identity by suppressing the venous fate in developing artery cells. In mice, the Notch4 receptor and the Delta-like 4 (Dll4) ligand are specifically expressed in arterial endothelial cells, suggesting a similar role. Here we show that the Dll4 ligand alone is required in a dosage-sensitive manner for normal arterial patterning in development. This implicates Dll4 as the specific mammalian endothelial ligand for autocrine endothelial Notch signaling, and suggests that Dll4 may be a suitable target for intervention in arterial angiogenesis.

Animals↗
PubMed2004

Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease.

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation-screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non-native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain- or loss-of-function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease-causing misfolded proteins. Rescue of misfolded, "trafficking-defective", but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or "pharmacoperones") are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native-type-like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin-releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.

Animals↗
PubMed2005

MaxiK channel partners: physiological impact.

The basic functional unit of the large-conductance, voltage- and Ca2+-activated K+ (MaxiK, BK, BKCa) channel is a tetramer of the pore-forming alpha-subunit (MaxiKalpha) encoded by a single gene, Slo, holding multiple alternative exons. Depending on the tissue, MaxiKalpha can associate with modulatory beta-subunits (beta1-beta4) increasing its functional diversity. As MaxiK senses and regulates membrane voltage and intracellular Ca2+, it links cell excitability with cell signalling and metabolism. Thus, MaxiK is a key regulator of vital body functions, like blood flow, uresis, immunity and neurotransmission. Epilepsy with paroxysmal dyskinesia syndrome has been recognized as a MaxiKalpha-related disorder caused by a gain-of-function C-terminus mutation. This channel region is also emerging as a key recognition module containing sequences for MaxiKalpha interaction with its surrounding signalling partners, and its targeting to cell-specific microdomains. The growing list of interacting proteins highlights the possibility that associations with the C-terminus of MaxiKalpha are dynamic and depending on each cellular environment. We speculate that the molecular multiplicity of the C-terminus (and intracellular loops) dictated by alternative exons may modulate or create additional interacting sites in a tissue-specific manner. A challenge is the dissection of MaxiK macromolecular signalling complexes in different tissues and their temporal association/dissociation according to the stimulus.

Animals↗
PubMed1990

Murine developmental control genes.

Various strategies have been used to isolate genes that participate in the regulation of mouse development. Gene families that have been identified on the basis of their homology to motifs within Drosophila control genes or human transcription factor genes, namely homeobox (Hox), paired-box (Pax), and POU genes, can be compared with respect to gene organization, structure, and expression patterns. The functions of these genes can be analyzed molecularly in vitro and in vivo with the use of available mouse mutants or transgenic mice. In addition, it has been possible to generate gain- or loss-of-function mutations by random or targeted introduction of transgenes. Models derived from these studies can reveal the successive steps of developmental control on a genetic level.

Animals↗
PubMed2002

Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships.

This review focuses on recent advances in the structure-function relationships of thyroid-stimulating hormone (TSH) and its receptor. TSH is a member of the glycoprotein hormone family constituting a subset of the cystine-knot growth factor superfamily. TSH is produced by the pituitary thyrotrophs and released to the circulation in a pulsatile manner. It stimulates thyroid functions using specific membrane TSH receptor (TSHR) that belongs to the superfamily of G protein-coupled receptors (GPCRs). New insights into the structure-function relationships of TSH permitted better understanding of the role of specific protein and carbohydrate domains in the synthesis, bioactivity, and clearance of this hormone. Recent progress in studies on TSHR as well as studies on the other GPCRs provided new clues regarding the molecular mechanisms of receptor activation. Such advances are a result of extensive site-directed mutagenesis, peptide and antibody approaches, detailed sequence analyses, and molecular modeling as well as studies on naturally occurring gain- and loss-of-function mutations. This review integrates expanding information on TSH and TSHR structure-function relationships and summarizes current concepts on ligand-dependent and -independent TSHR activation. Special emphasis has been placed on TSH domains involved in receptor recognition, constitutive activity of TSHR, new insights into the evolution of TSH bioactivity, and the development of high-affinity TSH analogs. Such structural, physiological, pathophysiological, evolutionary, and therapeutic implications of TSH-TSHR structure-function studies are frequently discussed in relation to concomitant progress made in studies on gonadotropins and their receptors.

Amino Acid Sequence↗
PubMed2005

Gain of von Willebrand factor-binding function by mutagenesis of a species-conserved residue within the leucine-rich repeat region of platelet glycoprotein Ibalpha.

Glycoprotein (GP) Ibalpha, a member of the leucine-rich repeat (LRR) protein family, mediates platelet adhesion to immobilized von Willebrand factor (VWF). We investigated the role in VWF binding of charged residues in the LRR region of GP Ibalpha that are conserved in human, canine, and murine proteins. Substitution of His86 with either Ala or Glu resulted in a gain of VWF-binding function as judged by increased VWF binding in the presence of the modulators ristocetin and botrocetin and by enhanced adhesion of Chinese hamster ovary (CHO) cells expressing the mutant GP Ibalpha to immobilized VWF under conditions of flow. This is the first report of a gain-of-function phenotype resulting from mutations in the LRR region of GP Ibalpha. Because His86 is 2 nm away from the region of GP Ibalpha with the largest surface of contact with VWF, the data suggest that the LRRs regulate GP Ibalpha affinity for VWF allosterically.

Allosteric Regulation↗
PubMed2003

Early activation of antioxidant mechanisms in muscle of mutant Cu/Zn-superoxide dismutase-linked amyotrophic lateral sclerosis mice.

A subset of familial ALS cases is associated with missense mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1), a free radical scavenging enzyme that protects cells against oxidative stress. Overexpression of these ALS-linked mutations confers an unidentified gain of function to the enzyme that triggers a series of neurological disorders characteristic of human ALS. To understand how skeletal muscle may counteract the progression of the disease, we explored the expression of different molecular effectors involved in antioxidant pathways. Our results are strongly indicative of the early and long-lasting activation of a series of molecular effectors thought to act coordinately in preventing the increased oxidative stress characteristic of ALS.

Aging↗
PubMed1999

Transition from monomeric to homodimeric DNA binding by nuclear receptors: identification of RevErbAalpha determinants required for RORalpha homodimer complex formation.

Nuclear hormone receptors belong to a class of transcription factors that recognize specific DNA sequences either as monomers, homodimers, or heterodimers with the common partner retinoic X receptor. In vitro mutagenesis studies, as well as determination of the crystal structure of several complexes formed by the DNA-binding domain of receptors bound to their cognate response elements, have begun to explain the molecular basis for protein-DNA and protein-protein interactions essential for high-affinity and specific DNA binding by nuclear receptors. In this study, we have used the related orphan nuclear receptors, RORalpha and RevErbAalpha, to study the molecular determinants involved in the transition from monomeric to homodimeric modes of DNA binding by nuclear receptors. While both receptors bind DNA as monomers to a response element containing a core AGGTCA half-site preceded by a 5'-A/T-rich flanking sequence, RevErbAalpha also binds as a homodimer to an extended DR2 element. Gain-of-function experiments using point mutations and subdomain swaps between RORalpha and RevErbAalpha identify four amino acids within RevErbAalpha sufficient to confer RORalpha with the ability to form cooperative homodimer complexes on an extended DR2. This study reveals how the transition from monomer to homodimer DNA binding by members of the nuclear receptor superfamily could be achieved from relatively few amino acid substitutions.

Amino Acid Sequence↗
PubMed2005

KIT-positive gastrointestinal stromal tumor in a 22-year-old male chimpanzee (Pan troglodites).

Gastrointestinal stromal tumors (GIST), KIT-positive and KIT signaling driven or platelet-derived growth factor receptor alpha (PDGFRA) signaling driven mesenchymal tumors, are poorly known in nonhuman primates. Availability of KIT- and PDGFRA-inhibitor drug imatinib mesylate has greatly raised the interest for these tumors. At necropsy of a 22-year-old male chimpanzee, a round, firm 2-cm intramural tumor was incidentally found in the midbody of the stomach and diagnosed as a GIST. Histologically, the mass was composed of spindle to polygonal epithelioid cells arranged in short to intermediate-length, interlacing streams, bundles, and nodular whorls often separated by hyalinized eosinophilic matrix. The mitotic rate was a maximum 1/50 high-power field. Immunohistochemically, the tumor cells were diffusely positive for KIT and CD34, focally positive for alpha-smooth muscle actin, and negative for muscle specific actin, desmin, S-100 protein, synaptophysin, and glial fibrillary acidic protein. Because the majority of human GISTs have gain-of-function KIT or PDGFRA mutations, genomic sequences of KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18 from this chimpanzee GIST were polymerase chain reaction amplified and sequenced. However, no mutation was identified in the analyzed "mutational hot spots." This study is the first extensive histomorphologic, immunohistochemical, and molecular genetic analysis of a chimpanzee GIST. More cases of nonhuman primate GISTs should be analyzed to discover the clinicopathologic spectrum of GISTs in these species.

Animals↗
PubMed2016

The Revolution in Migraine Genetics: From Aching Channels Disorders to a Next-Generation Medicine.

Channelopathies are a heterogeneous group of neurological disorders resulting from dysfunction of ion channels located in cell membranes and organelles. The clinical scenario is broad and symptoms such as generalized epilepsy (with or without fever), migraine (with or without aura), episodic ataxia and periodic muscle paralysis are some of the best known consequences of gain- or loss-of-function mutations in ion channels. We review the main clinical effects of ion channel mutations associated with a significant impact on migraine headache. Given the increasing and evolving use of genetic analysis in migraine research-greater emphasis is now placed on genetic markers of dysfunctional biological systems-we also show how novel information in rare monogenic forms of migraine might help to clarify the disease mechanisms in the general population of migraineurs. Next-generation sequencing (NGS) and more accurate and precise phenotyping strategies are expected to further increase understanding of migraine pathophysiology and genetics.

astrocyte↗
PubMed2006

The limitless role of p53 in cell cycle machinery: good news or bad news?

The p53 tumor suppressor gene acts as a great protagonist in deciding how cells undergo either cell cycle arrest or apoptosis after experiencing various stress signals, including DNA damage, hypoxia, oncogene activation, and hyperproliferation. Research on p53 is in steady expansion, as evidenced by the continual flood of papers claiming novel mutations, gain or loss of p53 functions, and gene interactions. The latest study carried out by Spurgers of Texas University and his Colleagues (J Biol Chem 2006; 281:25134-42) emphasizes the strong impact of p53 in the complicated machinery that regulates cell cycle progression. In this paper, microarray data and well-evaluated statistical procedures on PC3 and LNCaP prostate carcinoma cells, open new perspectives in p53 mechanisms and bring the simultaneous identification of novel p53-repressed cell cycle genes, hopefully providing significant improvements in the study of DNA damage response, multistep carcinogenesis, and treatment rationales and outcomes.

Animals↗
PubMed2002

The Ras1-mitogen-activated protein kinase signal transduction pathway regulates synaptic plasticity through fasciclin II-mediated cell adhesion.

Ras proteins are small GTPases with well known functions in cell proliferation and differentiation. In these processes, they play key roles as molecular switches that can trigger distinct signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide-3 kinase pathway, and the Ral-guanine nucleotide dissociation stimulator pathway. Several studies have implicated Ras proteins in the development and function of synapses, but the molecular mechanisms for this regulation are poorly understood. Here, we demonstrate that the Ras-MAPK pathway is involved in synaptic plasticity at the Drosophila larval neuromuscular junction. Both Ras1 and MAPK are expressed at the neuromuscular junction, and modification of their activity levels results in an altered number of synaptic boutons. Gain- or loss-of-function mutations in Ras1 and MAPK reveal that regulation of synapse structure by this signal transduction pathway is dependent on fasciclin II localization at synaptic boutons. These results provide evidence for a Ras-dependent signaling cascade that regulates fasciclin II-mediated cell adhesion at synaptic terminals during synapse growth.

Animals↗
PubMed1998

Structure and function of the Mec-ENaC family of ion channels.

The epithelial sodium channel (ENaC) is the prototype of a new family of ion channels known as the Mec-ENaC superfamily. This new family of proteins are involved in a wide variety of functions that range from maintenance of sodium homeostasis to transduction of mechanical stimuli and nociceptive pain by specialized neurons. They show distinct tissue- and cell type-dependent expression and differential sensitivity to inhibition by the diuretic amiloride and its analogs. Despite the very little amino acid identity shared by these proteins, they all have the same common structure that has become a hallmark of the Mec-ENaC superfamily. The efforts to understand the structure and regulation of these ion channels have been stimulated by the recent discovery of severe disturbances in the maintenance of blood pressure caused by gain- or loss-of-function mutations in the genes that encode the subunits of ENaC in humans. Moreover, cloning of the ion channels that mediate pain elicited by tissue injury and inflammation will facilitate the development of new drugs to treat these common ailments.

Epithelial Sodium Channels↗
PubMed2006

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells.

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.

Alkaline Phosphatase↗
PubMed1998

Presenilin mutations in Alzheimer's disease.

The presenilins (PS-1 and PS-2) are 2 members of a novel family of genes encoding integral membrane proteins recently implicated in Alzheimer's disease (AD) pathology. To date, 43 mutations have been identified in PS-1 and 2 in PS-2 that lead to familial presenile AD (onset before age 65 years). The normal and pathological functions of the PS proteins (ps-1 and ps-2) are unknown, but their high degree of homology predicts similar biological activities. Homologies with ps from other species suggest that they may play a role in intracellular protein sorting and trafficking, in intercellular cell signaling, or in cell death. Since to date only missense mutations and in-frame deletions were identified, it is believed that mutated ps act through either a gain of (dys-)function or a dominant negative effect. In vivo and in vitro studies have linked PS mutations to amyloid deposition, an early pathological event in AD brains.

Age of Onset↗
PubMed1998

Changes in auxin response from mutations in an AUX/IAA gene.

Transcription of the AUX/IAA family of genes is rapidly induced by the plant hormone auxin, but evidence that AUX/IAA genes mediate further responses to auxin has been elusive. Changes in diverse auxin responses result from mutations in the Arabidopsis AXR3 gene. AXR3 was shown to be a member of the AUX/IAA family, providing direct evidence that AUX/IAA genes are central in auxin signaling. Molecular characterization of axr3 gain-of-function and loss-of-function mutations established the functional importance of domains conserved among AUX/IAA proteins.

Amino Acid Sequence↗
PubMed2003

Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia.

We report here that individuals with Noonan syndrome and juvenile myelomonocytic leukemia (JMML) have germline mutations in PTPN11 and that somatic mutations in PTPN11 account for 34% of non-syndromic JMML. Furthermore, we found mutations in PTPN11 in a small percentage of individuals with myelodysplastic syndrome (MDS) and de novo acute myeloid leukemia (AML). Functional analyses documented that the two most common mutations in PTPN11 associated with JMML caused a gain of function.

Animals↗