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PubMed2006

Modulation of ethylene responses affects plant salt-stress responses.

Ethylene signaling plays important roles in multiple aspects of plant growth and development. Its functions in abiotic stress responses remain largely unknown. Here, we report that alteration of ethylene signaling affected plant salt-stress responses. A type II ethylene receptor homolog gene NTHK1 (Nicotiana tabacum histidine kinase 1) from tobacco (N. tabacum) conferred salt sensitivity in NTHK1-transgenic Arabidopsis (Arabidopsis thaliana) plants as judged from the phenotypic change, the relative electrolyte leakage, and the relative root growth under salt stress. Ethylene precursor 1-aminocyclopropane-1-carboxylic acid suppressed the salt-sensitive phenotype. Analysis of Arabidopsis ethylene receptor gain-of-function mutants further suggests that receptor function may lead to salt-sensitive responses. Mutation of EIN2, a central component in ethylene signaling, also results in salt sensitivity, suggesting that EIN2-mediated signaling is beneficial for plant salt tolerance. Overexpression of the NTHK1 gene or the receptor gain-of-function activated expression of salt-responsive genes AtERF4 and Cor6.6. In addition, the transgene NTHK1 mRNA was accumulated under salt stress, suggesting a posttranscriptional regulatory mechanism. These findings imply that ethylene signaling may be required for plant salt tolerance.

Arabidopsis↗
PubMed2002

A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion.

Potassium ions (K(+)) are the most abundant cations in plants and are necessary for cell growth. Arabidopsis shy3-1 mutant plants have a short hypocotyl, small leaves, and a short flowering stem, and these defects result from decreased cell expansion. The semidominant shy3-1 mutation changes an amino acid in KT2/KUP2, a K(+) transporter related to the Escherichia coli Kup protein. Second mutations in the KT2/KUP2/SHY3 gene, including presumed null mutations, suppress the shy3-1 phenotypes. Plants with these intragenic suppressor mutations appear similar to wild-type plants, suggesting that KT2/KUP2/SHY3 acts redundantly with other genes. Expression of the shy3-1 mutant version of KT2/KUP2/SHY3 in wild-type plants confers shy3-1-like phenotypes, indicating that shy3-1 probably either causes a gain of function or creates an interfering protein. The shy3-1 mutation does not eliminate the ability of the KT2/KUP2 cDNA to rescue the growth of a potassium transport-deficient E. coli mutant. A P(SHY3)::GUS fusion is expressed in growing portions of the plant. These results suggest that KT2/KUP2/SHY3 mediates K(+)-dependent cell expansion in growing tissues.

Arabidopsis↗
PubMed1997

Residues of the cytoplasmic domain of MotA essential for torque generation in the bacterial flagellar motor.

The MotA protein of Escherichia coli is a component of the flagellum that functions, together with MotB, in transmembrane proton conduction. MotA and MotB are believed to form the stator of the flagellar motor. They are integral membrane proteins; MotA has a large (ca 22 kDa) domain in the cytoplasm, and MotB a much smaller one (ca 3 kDa). Recent work suggests that cytoplasmically located parts of MotA and/or MotB might be present at the active site for torque generation in the motor. To test the proposal that the cytoplasmic domain of MotA functions in torque generation, and to identify the amino acid residues most important for function, we have carried out a mutational analysis of this domain. Using random mutagenesis, many mutations of cytoplasmic residues of MotA were isolated, which either abolish or impair torque generation. In most cases the residues affected are not conserved, and many of the replacements involve loss or gain of a proline residue, which suggests that these mutations disrupt function by altering the protein conformation rather than by directly affecting residues of an active site. Using site-directed mutagenesis, the conserved residues in the cytoplasmic domain of MotA were replaced, either singly or, in the case of charged residues, in various combinations. The results identify four residues of MotA that are important for motor function. These are Arg90 and Glu98, located in the cytoplasmic domain, and Pro173 and Pro222, located at the interface between the cytoplasmic domain and the membrane-spanning domain. Possible roles for these residues in torque generation are discussed.

Amino Acid Sequence↗
PubMed1998

Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.

Point mutations in the presenilin-1 gene (PS1) are a major cause of familial Alzheimer's disease. They result in a selective increase in the production of the amyloidogenic peptide amyloid-beta(1-42) by proteolytic processing of the amyloid precursor protein (APP). Here we investigate whether PS1 is also involved in normal APP processing in neuronal cultures derived from PS1-deficient mouse embryos. Cleavage by alpha- and beta-secretase of the extracellular domain of APP was not affected by the absence of PS1, whereas cleavage by gamma-secretase of the transmembrane domain of APP was prevented, causing carboxyl-terminal fragments of APP to accumulate and a fivefold drop in the production of amyloid peptide. Pulse-chase experiments indicated that PS1 deficiency specifically decreased the turnover of the membrane-associated fragments of APP. As in the regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, PS1 appears to facilitate a proteolytic activity that cleaves the integral membrane domain of APP. Our results indicate that mutations in PS1 that manifest clinically cause a gain of function and that inhibition of PS1 activity is a potential target for anti-amyloidogenic therapy in Alzheimer's disease.

Amyloid Precursor Protein Secretases↗
PubMed2004

Sudden death in familial polymorphic ventricular tachycardia associated with calcium release channel (ryanodine receptor) leak.

BACKGROUND: Familial polymorphic ventricular tachycardia (FPVT) is characterized by exercise-induced arrhythmias and sudden cardiac death due to missense mutations in the cardiac ryanodine receptor (RyR2), an intracellular Ca2+ release channel required for excitation-contraction coupling in the heart. METHODS AND RESULTS: Three RyR2 missense mutations, P2328S, Q4201R, and V4653F, which occur in Finnish families, result in similar mortality rates of approximately 33% by age 35 years and a threshold heart rate of 130 bpm, above which exercise induces ventricular arrhythmias. Exercise activates the sympathetic nervous system, increasing cardiac performance as part of the fight-or-flight stress response. We simulated the effects of exercise on mutant RyR2 channels using protein kinase A (PKA) phosphorylation. All 3 RyR2 mutations exhibited decreased binding of calstabin2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. After PKA phosphorylation, FPVT-mutant RyR2 channels showed a significant gain-of-function defect consistent with leaky Ca2+ release channels and a significant rightward shift in the half-maximal inhibitory Mg2+ concentration (IC50). Treatment with the experimental drug JTV519 enhanced binding of calstabin2 to RyR2 and normalized channel function. CONCLUSIONS: Sympathetic activation during exercise induces ventricular arrhythmias above a threshold heart rate in RyR2 mutation carriers. Simulating the downstream effects of the sympathetic activation by PKA phosphorylation of RyR2 channels containing these FPVT missense mutations produced a consistent gain-of-function defect. RyR2 function and calstabin2 depletion were rescued by JTV519, suggesting stabilization of the RyR2 channel complex may represent a molecular target for the treatment and prevention of exercise-induced arrhythmias and sudden death in these patients.

Adult↗
PubMed2025

Comparative Genomic Analysis of Six Mycoplasma Gallisepticum Strains: Insights into Genetic Diversity and Antibiotic Resistance.

Mycoplasma gallisepticum (MG) is a significant pathogen that causes respiratory diseases, which have had a substantial economic impact on the poultry industry. Despite the resistance of MG to antibiotics, it is imperative to identify genetic diversity in order to develop countermeasures. In this study, the genomes of six MG strains were examined to gain deeper insights into the mutations. The data pertaining to Variant Annotation and Mutation Analysis using SnpEff, along with the calculation of mutation rates as the ratio of total mutations to the length of the genomic regions analyzed, were thoroughly examined. The comprehensive evaluation yielded a total of 25,942 variants across the six strains, underscoring substantial genetic diversity. Notably, strain S6 exhibited a preponderance of frameshift mutations. A notable finding was the presence of a mutation in the MsbA gene shared by all six strains. Furthermore, five of the six strains, with the exception of strain F99 Lab, exhibited a mutation at position 5158, which impacts a multidrug transport system. Notably, strain ATCC exhibits a distinctive mutation at position 942, while strain S6 displays a unique mutation at position 6855, which is linked to efflux ABC transporter components. Furthermore, a substantial degree of genetic variation was observed among the CrmA, GapA, and vlhA genes among the various strains. High-impact changes, such as insertions and deletions, exhibited a higher frequency in CrmA, particularly in strain S6. Conversely, nonsynonymous variations demonstrated a heightened prevalence in GapA, particularly in strain F99 Lab. The vlhA gene exhibited a spectrum of effects, ranging from synonymous mutations to high-impact mutations such as stop-gains and frameshifts, particularly in strains k5111a and k4602. The functional variations observed among the strains can be attributed to these mutations, which have the potential to alter gene expression or protein function. Furthermore, substantial mutations in the dxr and rpoC genes were associated with antibiotic resistance. These mutations underscore the ongoing evolutionary adaptations of M. gallisepticum. Consequently, there is an imperative for the revision of treatment protocols and the formulation of targeted vaccines to regulate resistance within the poultry industry.

Mycoplasma gallisepticum↗
PubMed2006

A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation.

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

Animals↗
PubMed2003

Differentially expressed downstream genes in cells with normal or mutated p53.

Mutations in p53 gene could lead to loss of function, negative complementation, or to gain-of-oncogenic functions, thus leading to the increase of tumorigenicity and invasiveness of cancer. This study focused on cancer-related p53 mutants, including A138T, C141Y, RI58L, G245C, and R248Q. Using a modified differential display technique, response profiles of plasmid-expressed wild-type as well as mutated p53, in comparison to p53-null cells, are being established. These profiles can help our understanding of p53 involvement in cancer, leading to accurate diagnosis, treatment, and prognostic analysis by: 1) comprehensive knowledge of p53 network gene expression profiles; 2) finding the most significant gene expression profiles of p53 mutants; 3) revealing genes that only respond to p53 mutants (gain of function). Our results showed significant differences in the expression patterns among p53-null. wild-type p53, and p53 mutants A138T, C141Y, R158L, G245C, and R248Q samples. We also report here the first found p53 mutant-triggered alternative splicing.

Alternative Splicing↗
PubMed1994

Hot-spot p53 mutants interact specifically with two cellular proteins during progression of the cell cycle.

Inactivation of both alleles of the p53 gene is commonly found in human cancers. In contrast to mutations of the retinoblastoma gene, certain altered forms of p53 gain growth-promoting functions. To explore the mechanisms underlying this gain of function, we have identified two nuclear proteins, with molecular masses of 42 and 38 kDa, respectively, that are specifically associated with p53 mutated within the simian virus 40 T-antigen-binding domain, "hot spots" found in many human tumors. These mutants transactivate the multiple-drug resistance gene promoter and cause cells to grow to higher density. Both the mutated p53 complex with p42 and p38 increase when cells enter S phase of the cell cycle but decrease in G1 and M phases, suggesting that they may have a role in promoting cell growth.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Protein folding revisited. A polypeptide chain at the folding-misfolding-nonfolding cross-roads: which way to go?

The structure-function paradigm claims that a specific function of a protein is determined by its unique and rigid three-dimensional (3D) structure. Thus, following its biosynthesis on the ribosome, a protein must fold to be functional. This idea represents one of the cornerstones of modern biology. Numerous cases when, due to the effect of environmental factors or because of genetic defects (mutations), a polypeptide chain has lost its capability to gain a proper functional 3D structure (i.e. became misfolded), seem to confirm this concept. Consequences of such misfolding are well known and represent lost of function, aggregation, development of conformational disorders and cell death. However, the recent revelation of countless examples of intrinsically disordered proteins has cast doubt on the general validity of the structure-function paradigm and revealed an intriguing route of functional disorder. Thus, in a living cell, a polypeptide chain chooses between three potential fates - functional folding, potentially deadly misfolding and mysterious nonfolding. This choice is dictated by the peculiarities of amino acid sequence and/or by the pressure of environmental factors. The aim of the present review is to outline some interesting features of these three routes.

Amino Acid Sequence↗
PubMed1997

Oxidative stress mediates impairment of muscle function in transgenic mice with elevated level of wild-type Cu/Zn superoxide dismutase.

Cases of familial amyotrophic lateral sclerosis (fALS; a neurodegenerative disorder) have been reported in which the gene for Cu/Zn superoxide dismutase (CuZnSOD) was mutated. Several studies with the fALS mutant CuZnSOD in transgenic mice and cells showed that the fALS mutations act through an as yet undefined dominant gain-of-function mechanism. Wild-type CuZnSOD catalyzes the dismutation of superoxide (O(2)(-).) but also produces hydroxyl radicals (.OH) with H(2)O(2) as substrate. Two laboratories have recently demonstrated that the .OH production ability was preferentially enhanced by the fALS mutant CuZnSOD, suggesting that this might be the function gained in fALS. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of wild-type CuZnSOD was also associated with increased .OH production and impaired muscle function. Enhanced formation of .OH was detected, by spin trapping, in brain and muscle extracts of the Tg-CuZnSOD mice. Three independently derived Tg-CuZnSOD lines showed muscle abnormalities, reflected by altered electromyography (EMG) and diminished performance in the rope grip test. After treatment with paraquat (PQ), a widely used herbicide and O(2)(-).-generating compound, muscle disability significantly deteriorated in Tg-CuZnSOD mice but not in control mice. The results indicate that elevated levels of CuZnSOD cause indigenous long-term oxidative stress leading to impairment of muscle function. These findings may provide valuable clues about the concurred role of indigenous oxidative stress and exogenous agents in the etiology of sporadic ALS and several other neurodegenerative diseases in which a specific subset of neurons is affected.

Animals↗
PubMed2003

Amino acid substitutions in VP2 residues contacting sialic acid in low-neurovirulence BeAn virus dramatically reduce viral binding and spread of infection.

Theiler's murine encephalomyelitis viruses (TMEV) consist of two groups, the high- and low-neurovirulence groups, based on lethality in intracerebrally inoculated mice. Low-neurovirulence TMEV result in a persistent central nervous system infection in mice, leading to an inflammatory demyelinating pathology and disease. Low- but not high-neurovirulence strains use sialic acid as an attachment factor. The recent resolution of the crystal structure of the low-neurovirulence DA virus in complex with the sialic acid mimic sialyllactose demonstrated that four capsid residues make contact with sialic acid through noncovalent hydrogen bonds. To systematically test the importance of these sialic acid-binding residues in viral entry and infection, we mutated three VP2 puff B amino acids proposed to make contact with sialic acid and analyzed the consequences of each amino acid substitution on viral entry and spread. The fourth residue is in the VP3-VP1 cleavage dipeptide and could not be mutated. Our data suggest that residues Q2161 and G2174 are directly involved in BeAn virus attachment to sialic acid and that substitutions of these two residues result in the loss of or reduced viral binding and hemagglutination and in the inability to spread among BHK-21 cells. In addition, a gain of function-revertant virus was recovered with the Q2161A mutation after prolonged passage in cells.

Amino Acid Substitution↗
PubMed2002

Lack of Bcl10 gene mutations in laryngeal squamous cell carcinoma.

The Bcl10 gene encodes a protein probably involved in some apoptotic regulatory pathways. Bcl10 mutations lead to the translation of truncated proteins that show gain-of-function transforming activity; it has been suggested that Bcl10 may represent a major target gene for inactivation in many human cancers. To define the frequency of Bcl10 mutations in laryngeal squamous cell carcinoma and their possible association with tumour progression, we investigated a large panel of tumours representative of all grades and stages of malignancy. To detect pathogenic mutations in exons 1, 2 and 3 of the Bcl10 gene, we performed a silver-staining polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis followed by direct DNA sequencing. We revealed the presence of SSCP variants in 18 out of 91 laryngeal tumours. Direct DNA sequencing showed previously described polymorphisms but no pathogenic mutations. We have strong evidence that the Bcl10 gene is not involved in laryngeal carcinogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2004

A patch of surface-exposed residues mediates negative regulation of immune signaling by tomato Pto kinase.

Tomato (Lycopersicon esculentum) Pto kinase specifically recognizes the Pseudomonas effector proteins AvrPto and AvrPtoB, leading to induction of defense responses and hypersensitive cell death. Structural modeling of Pto combined with site-directed mutagenesis identified a patch of surface-exposed residues required for native regulation of signaling. Mutations in this area resulted in constitutive gain-of-function (CGF) forms of Pto that activated AvrPto-independent cell death via the cognate signaling pathway. The patch overlaps the peptide binding region of the kinase catalytic cleft and is part of a broader region required for interaction with bacterial effectors. We propose that the negative regulatory patch is normally occupied by a peptide that represses Pto signaling. Furthermore, we found that Pto kinase activity was required for Avr-dependent activation but dispensable for signaling by CGF forms of Pto. This suggests that Pto signals by a conformational change rather than phosphorylation of downstream substrates in the defense signaling pathway.

Amino Acids↗
PubMed2002

Molecular basis of an inherited epilepsy.

Epilepsy is a common neurological condition that reflects neuronal hyperexcitability arising from largely unknown cellular and molecular mechanisms. In generalized epilepsy with febrile seizures plus, an autosomal dominant epilepsy syndrome, mutations in three genes coding for voltage-gated sodium channel alpha or beta1 subunits (SCN1A, SCN2A, SCN1B) and one GABA receptor subunit gene (GABRG2) have been identified. Here, we characterize the functional effects of three mutations in the human neuronal sodium channel alpha subunit SCN1A by heterologous expression with its known accessory subunits, beta1 and beta2, in cultured mammalian cells. SCN1A mutations alter channel inactivation, resulting in persistent inward sodium current. This gain-of-function abnormality will likely enhance excitability of neuronal membranes by causing prolonged membrane depolarization, a plausible underlying biophysical mechanism responsible for this inherited human epilepsy.

Cells, Cultured↗
PubMed1997

Transgenic models of Huntington's disease.

CAG/polyglutamine expansion has been shown to form the molecular basis of an increasing number of inherited neurodegenerative diseases. The mutation is likely to act by a dominant gain of function but the mechanism by which it leads to neuronal dysfunction and cell death is unknown. The proteins harbouring these polyglutamine tracts are unrelated and without exception are widely expressed with extensively overlapping expression patterns. The factors governing the cell specific nature of the neurodegeneration have yet to be understood. Upon a certain size threshold, expanded CAG repeats become unstable on transmission and a modest degree of somatic mosaicism is apparent. Similarly, the molecular basis of the instability and its tissue specificity has yet to be unravelled. Recent reports describing the first mouse models of CAG/polyglutamine disorders indicate that it will be possible to model both the pathogenic mechanism and the CAG repeat instability in the mouse. This has great potential and promise for uncovering the molecular basis of these diseases and developing therapeutic interventions.

Animals↗
PubMed1996

Spinal and bulbar muscular atrophy: androgen receptor dysfunction caused by a trinucleotide repeat expansion.

Kennedy's disease (spinal and bulbar muscular atrophy) is an X-linked form of motor neuron disease that affects adult men. The syndrome is characterized by progressive atrophy of the limb muscles, pelvic and shoulder girdles and dysphagia and dysarthria, and is caused by the degeneration of spinal and bulbar motor neurons. Kennedy's disease is caused by a trinucleotide repeat expansion of a CAG repeat in exon A of the androgen receptor gene, and is one of a group of neurological diseases caused by trinucleotide repeat expansions in different genes. The mutation in Kennedy's disease involves an increased number of glutamine residues in the amino-terminal domain of the receptor. Point mutations and deletions in the androgen receptor gene cause androgen insensitivity syndrome, however subjects with Kennedy's disease have normal virilization, although progressive gynaecomastia, testicular atrophy and infertility may occur. Androgen receptors are expressed widely in the normal brain, and in the anterior horn cells of the spinal cord; however, their role in neuronal tissue is not known, nor is it known how the androgen receptor gene mutation causes neuronal degeneration. Kennedy's disease is likely to be a 'gain of function' abnormality, so that the presence of the receptor with an increased number of glutamines is toxic to motor neurons. It is possible that the mutation alters interaction of the receptor with other neuronal transcription factors, or neuronotoxicity may occur because of a non-specific effect caused by the presence of a protein with a large homoglutamine domain. Studies of patients with Kennedy's disease have shown that expression of androgen receptor mRNA and protein in spinal cord may be decreased, as can be the affinity of the mutant receptor for androgen. In vitro studies have shown impaired transcription activation ability of the mutant androgen receptor. The age at onset of Kennedy's disease may correlate with the size of the CAG repeat, however there is a large degree of variability of age at onset between subjects with the same number of repeats. Further study of the effect of the Kennedy's disease mutation on androgen receptor function in motor neurons will allow us to increase our understanding of the pathogenesis of this disease.

Animals↗
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↗