Functional analysis of mutant proteins in cells

2005 ◽  
pp. 191-196
Keyword(s):  
Functional Analysis ◽  
Mutant Proteins ◽  
In Cells

scholarly journals Functional analysis of the Notch ligand Jagged1 missense mutant proteins underlying Alagille syndrome

FEBS Journal ◽  
2012 ◽  
Vol 279 (12) ◽  
pp. 2096-2107 ◽  
Author(s):  
Minoru Tada ◽  
Satsuki Itoh ◽  
Akiko Ishii-Watabe ◽  
Takuo Suzuki ◽  
Nana Kawasaki
Keyword(s):  
Functional Analysis ◽  
Alagille Syndrome ◽  
Notch Ligand ◽  
Mutant Proteins ◽  
Missense Mutant

scholarly journals Herpes Simplex Virus Type 1 Portal Protein UL6 Interacts with the Putative Terminase Subunits UL15 and UL28

2003 ◽  
Vol 77 (11) ◽  
pp. 6351-6358 ◽  
Author(s):  
Colleen A. White ◽  
Nigel D. Stow ◽  
Arvind H. Patel ◽  
Michelle Hughes ◽  
Valerie G. Preston
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Dna Packaging ◽  
Mutant Proteins ◽  
Simplex Virus ◽  
In Cells ◽  
Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL6, UL15, and UL28 proteins are essential for cleavage of replicated concatemeric viral DNA into unit length genomes and their packaging into a preformed icosahedral capsid known as the procapsid. The capsid-associated UL6 DNA-packaging protein is located at a single vertex and is thought to form the portal through which the genome enters the procapsid. The UL15 protein interacts with the UL28 protein, and both are strong candidates for subunits of the viral terminase, a key component of the molecular motor that drives the DNA into the capsid. To investigate the association of the UL6 protein with the UL15 and UL28 proteins, the three proteins were produced in large amounts in insect cells with the baculovirus expression system. Interactions between UL6 and UL28 and between UL6 and UL15 were identified by an immunoprecipitation assay. These results were confirmed by transiently expressing wild-type and mutant proteins in mammalian cells and monitoring their distribution by immunofluorescence. In cells expressing the single proteins, UL6 and UL15 were concentrated in the nuclei whereas UL28 was found in the cytoplasm. When the UL6 and UL28 proteins were coexpressed, UL28 was redistributed to the nuclei, where it colocalized with UL6. In cells producing either of two cytoplasmic UL6 mutant proteins and a functional epitope-tagged form of UL15, the UL15 protein was concentrated with the mutant UL6 protein in the cytoplasm. These observed interactions of UL6 with UL15 and UL28 are likely to be of major importance in establishing a functional DNA-packaging complex at the portal vertex of the HSV-1 capsid.

scholarly journals Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen

2021 ◽  
Vol 14 (1) ◽  
pp. 11-33
Author(s):  
Sui Sawaguchi ◽  
Kenji Tago ◽  
Hiroaki Oizumi ◽  
Katsuya Ohbuchi ◽  
Masahiro Yamamoto ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Mtor Signaling ◽  
Marker Protein ◽  
Wild Type ◽  
Mutant Proteins ◽  
Oligodendroglial Cell ◽  
Hypomyelinating Leukodystrophy ◽  
In Cells

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD−102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.

scholarly journals Rare Neurologic Disease-Associated Mutations of AIMP1 Are Related with Inhibitory Neuronal Differentiation Which Is Reversed by Ibuprofen

Medicines ◽  
2020 ◽  
Vol 7 (5) ◽  
pp. 25
Author(s):  
Yu Takeuchi ◽  
Marina Tanaka ◽  
Nanako Okura ◽  
Yasuyuki Fukui ◽  
Ko Noguchi ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Cell Model ◽  
Neuronal Cell ◽  
Fiber Formation ◽  
Congenital Diseases ◽  
Mutant Proteins ◽  
Frame Shift ◽  
Actin Fiber ◽  
In Cells

Background: Hypomyelinating leukodystrophy 3 (HLD3), previously characterized as a congenital diseases associated with oligodendrocyte myelination, is increasingly regarded as primarily affecting neuronal cells. Methods: We used N1E-115 cells as the neuronal cell model to investigate whether HLD3-associated mutant proteins of cytoplasmic aminoacyl-tRNA synthase complex-interacting multifunctional protein 1 (AIMP1) aggregate in organelles and affect neuronal differentiation. Results: 292CA frame-shift type mutant proteins harboring a two-base (CA) deletion at the 292th nucleotide are mainly localized in the lysosome where they form aggregates. Similar results are observed in mutant proteins harboring the Gln39-to-Ter (Q39X) mutation. Interestingly, the frame-shift mutant-specific peptide specifically interacts with actin to block actin fiber formation. The presence of actin with 292CA mutant proteins, but not with wild type or Q39X ones, in the lysosome is detectable by immunoprecipitation of the lysosome. Furthermore, expression of 292CA or Q39X mutants in cells inhibits neuronal differentiation. Treatment with ibuprofen reverses mutant-mediated inhibitory differentiation as well as the localization in the lysosome. Conclusions: These results not only explain the cell pathological mechanisms inhibiting phenotype differentiation in cells expressing HLD3-associated mutants but also identify the first chemical that restores such cells in vitro.

scholarly journals The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis

2000 ◽  
Vol 20 (19) ◽  
pp. 7146-7159 ◽  
Author(s):  
Dick D. Mosser ◽  
Antoine W. Caron ◽  
Lucie Bourget ◽  
Anatoli B. Meriin ◽  
Michael Y. Sherman ◽  
...  
Keyword(s):  
Protective Effects ◽  
Atpase Domain ◽  
Caspase Activation ◽  
Apoptotic Pathway ◽  
Mutant Proteins ◽  
Chaperone Function ◽  
Induced Apoptosis ◽  
Jnk Activation ◽  
In Cells

ABSTRACT Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochromec-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.

Expression and functional analysis of nucleic acid sensors in cells of the central nervous system

2014 ◽  
Vol 275 (1-2) ◽  
pp. 83
Author(s):  
Donal Cox ◽  
Glyn Williams ◽  
Neil Kearney ◽  
Andrew Bowie ◽  
Marina Lynch ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Functional Analysis ◽  
Nervous System ◽  
Nucleic Acid ◽  
The Central Nervous System ◽  
In Cells ◽  
Acid Sensors

Functional analysis of CFTR chloride channel activity in cells with elevated MDR1 expression

2003 ◽  
Vol 304 (2) ◽  
pp. 248-252 ◽  
Author(s):  
Lishuang Cao ◽  
Grzegorz Owsianik ◽  
Martine Jaspers ◽  
Annelies Janssens ◽  
Harry Cuppens ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Chloride Channel ◽  
Channel Activity ◽  
Mdr1 Expression ◽  
In Cells

Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP

1991 ◽  
Vol 11 (10) ◽  
pp. 4822-4829
Author(s):  
C L Farnsworth ◽  
L A Feig
Keyword(s):  
Double Mutant ◽  
Hydroxyl Group ◽  
Guanine Nucleotide ◽  
Structural Studies ◽  
Inhibitory Protein ◽  
Mutant Proteins ◽  
Downstream Target ◽  
Autophosphorylation Site ◽  
In Cells

We have previously demonstrated that substitution of Asn for Ser at position 17 of RasH yields a dominant inhibitory protein whose expression in cells interferes with endogenous Ras function (L. A. Feig, and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988). Subsequent structural studies have shown that the hydroxyl group of Ser-17 contributes to the binding of Mg2+ associated with bound nucleotide. In this report, we show that more subtle amino acid substitutions at this site that would be expected to interfere with complexing Mg2+, such as Cys or Ala, also generated dominant inhibitory mutants. In contrast, a Thr substitution that conserves a reactive hydroxyl group maintained normal Ras function. These results argue that the defect responsible for the inhibitory activity is improper coordination of Mg2+. Preferential affinity for GDP, observed in the original Asn-17 mutant, was found exclusively in inhibitory mutants. However, this binding specificity did not completely block the mutant proteins from binding GTP in vivo since introduction of the autophosphorylation site, Thr-59, in 17N Ras resulted in the phosphorylation of the double mutant in cells. Furthermore, inhibitory mutants failed to activate a model downstream target, yeast adenylate cyclase, even when bound to GTP. Thus, the consequence of improper complexing of Mg2+ was to lock the protein in a constitutively inactive state. A model is presented to explain how these properties could cause the mutant protein to inhibit the activation of endogenous Ras by competing for a guanine nucleotide-releasing factor.

scholarly journals Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (5) ◽  
pp. 2566-2575 ◽  
Author(s):  
T E Blank ◽  
M P Woods ◽  
C M Lebo ◽  
P Xin ◽  
J E Hopper
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Strong Support ◽  
Constitutive Expression ◽  
Mutant Proteins ◽  
Binding Characteristics ◽  
Isolation And Characterization ◽  
In Cells

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

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