scholarly journals An in vitro and computational validation of a novel loss-of-functional mutation in PAX9 associated with non-syndromic tooth agenesis

2022 ◽  
Author(s):  
Tanmoy Sarkara ◽  
Prashant Ranjan ◽  
Smitha Kanathur ◽  
Ankush Gupta ◽  
PARIMAL DAS
Keyword(s):  
Amino Acid Position ◽  
Binding Activity ◽  
Mutant Protein ◽  
Tooth Agenesis ◽  
Craniofacial Anomalies ◽  
Loss Of Function ◽  
Wild Type ◽  
Paired Domain ◽  
Computational Validation

Congenital tooth agenesis (CTA) is one of the most common craniofacial anomalies. Its frequency varies among different population depending upon the genetic heterogeneity. CTA could be of familial or sporadic and syndromic or non-syndromic. Five major genes are found to be associated with non-syndromic CTA namely, PAX9, MSX1, EDA1, AXIN2 and WNT10A. In this study, an India family with CTA was investigated and a novel c.336C>G variation was identified in the exon 3 of PAX9, leading to substitution of evolutionary conserved Cys with Trp at 112 amino acid position located at the functionally significant DNA binding paired domain region. Functional analysis revealed that p.Cys112Trp mutation did not prevent the nuclear localization although mutant protein had higher cytoplasmic retention. EMSA using e5 probe revealed that mutant protein was unable to bind with the paired-domain binding site. Subsequently, GST pull-down assay revealed lower binding activity of the mutant protein with its known interactor MSX1. Further RNA-sequencing of PAX9 over-expressed HEK293, identified two potential novel targets, WNT4 and WNT7b those are up-regulated by wild-type PAX9 but not by mutant. These in vitro results were consistent with the computational results. The in vitro and computational observations altogether suggest that c.336C>G (p.Cys112Trp) variation leads to loss-of-function of PAX9 leading to CTA in this family.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

2019 ◽  
Vol 116 (50) ◽  
pp. 25322-25328 ◽  
Author(s):  
Yi Liu ◽  
Xiaopin Ma ◽  
Hisashi Fujioka ◽  
Jun Liu ◽  
Shengdi Chen ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Cellular Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Efflux ◽  
And Function ◽  
The Brain ◽  
Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

scholarly journals A Polyamine Oxidase from Selaginella lepidophylla (SelPAO5) can Replace AtPAO5 in Arabidopsis through Converting Thermospermine to Norspermidine instead to Spermidine

Plants ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 99 ◽  
Author(s):  
G. H. M. Sagor ◽  
Tomonobu Kusano ◽  
Thomas Berberich
Keyword(s):  
Normal Values ◽  
Normal Growth ◽  
Polyamine Oxidase ◽  
Loss Of Function ◽  
Wild Type ◽  
In Planta ◽  
Selaginella Lepidophylla ◽  
Growth Phenotype ◽  
Polyamine Oxidases

Of the five polyamine oxidases in Arabidopsis thaliana, AtPAO5 has a substrate preference for the tetraamine thermospermine (T-Spm) which is converted to triamine spermidine (Spd) in a back-conversion reaction in vitro. A homologue of AtPAO5 from the lycophyte Selaginella lepidophylla (SelPAO5) back-converts T-Spm to the uncommon polyamine norspermidine (NorSpd) instead of Spd. An Atpao5 loss-of-function mutant shows a strong reduced growth phenotype when growing on a T-Spm containing medium. When SelPAO5 was expressed in the Atpao5 mutant, T-Spm level decreased to almost normal values of wild type plants, and NorSpd was produced. Furthermore the reduced growth phenotype was cured by the expression of SelPAO5. Thus, a NorSpd synthesis pathway by PAO reaction and T-Spm as substrate was demonstrated in planta and the assumption that a balanced T-Spm homeostasis is needed for normal growth was strengthened.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Peptide Deformylase in Staphylococcus aureus: Resistance to Inhibition Is Mediated by Mutations in the Formyltransferase Gene

2000 ◽  
Vol 44 (7) ◽  
pp. 1825-1831 ◽  
Author(s):  
Peter S. Margolis ◽  
Corinne J. Hackbarth ◽  
Dennis C. Young ◽  
Wen Wang ◽  
Dawn Chen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genomic Library ◽  
Specific Inhibitor ◽  
Peptide Deformylase ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Resistant Mutants

ABSTRACT Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs, defA and defB, were identified inStaphylococcus aureus. The defA homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR. A distinct homolog, defB, was cloned from an S. aureus genomic library by complementation of the arabinose-dependent phenotype of a P BAD -def Escherichia coli strain grown under arabinose-limiting conditions. Overexpression in E. coli of defB, but not defA, correlated to increased deformylase activity and decreased susceptibility to actinonin, a deformylase-specific inhibitor. ThedefB gene could not be disrupted in wild-type S. aureus, suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, thedefA gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not show cross-resistance to other classes of antibiotics. When compared to the parent, an actinonin-resistant strain produced an attenuated infection in a murine abscess model, indicating that this strain also has a growth disadvantage in vivo. Sequence analysis of the actinonin-resistant mutants revealed that each harbors a loss-of-function mutation in the fmt gene. Susceptibility to actinonin was restored when the wild-type fmt gene was introduced into these mutant strains. An S. aureusΔfmt strain was also resistant to actinonin, suggesting that a functional deformylase activity is not required in a strain that lacks formyltransferase activity. Accordingly, thedefB gene could be disrupted in an fmt mutant.

Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-β-induced nuclear accumulation

2003 ◽  
Vol 285 (4) ◽  
pp. C823-C830 ◽  
Author(s):  
Bernard A. J. Roelen ◽  
Ori S. Cohen ◽  
Malay K. Raychowdhury ◽  
Deborah N. Chadee ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Phosphorylation Site ◽  
Transforming Growth Factor Β ◽  
Nuclear Accumulation ◽  
Mutant Protein ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Smad4, the common Smad, is central for transforming growth factor (TGF)-β superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-β and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-β-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.

scholarly journals The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.

1994 ◽  
Vol 14 (2) ◽  
pp. 1333-1346 ◽  
Author(s):  
S Obert ◽  
R J O'Connor ◽  
S Schmid ◽  
P Hearing
Keyword(s):  
Binding Activity ◽  
Mutant Protein ◽  
Stable Complex ◽  
Physical Interaction ◽  
Dimerization Domain ◽  
Fab Fragment ◽  
Dna Binding Activity ◽  
Transcription Factor E2f

Binding of the mammalian transcription factor E2F to the adenovirus E2a early promoter is modulated through interaction with the viral E4-6/7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally, and E4-6/7 mutant with a phenylalanine-to-proline substitution at amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP-1, and E4-6/7 are sufficient to form the induced E2F complex at the E2a promoter.

scholarly journals Identification of a putative DNA-binding protein in Arabidopsis that acts as a susceptibility hub and interacts with multiple Pseudomonas syringae effectors

2020 ◽  
Author(s):  
Karl Schreiber ◽  
Jennifer D Lewis
Keyword(s):  
Pseudomonas Syringae ◽  
Transcriptional Profiling ◽  
Effector Proteins ◽  
Ribosomal Protein Genes ◽  
Loss Of Function ◽  
Wild Type ◽  
In Planta ◽  
Arabidopsis Protein ◽  
Wide Range

Phytopathogens use secreted effector proteins to suppress host immunity and promote pathogen virulence, and there is increasing evidence that the host-pathogen interactome comprises a complex network. In an effort to identify novel interactors of the Pseudomonas syringae effector HopZ1a, we performed a yeast two-hybrid screen that identified a previously uncharacterized Arabidopsis protein that we designate HopZ1a Interactor 1 (ZIN1). Additional analyses in yeast and in planta revealed that ZIN1 also interacts with several other P. syringae effectors. We show that an Arabidopsis loss-of-function zin1 mutant is less susceptible to infection by certain strains of P. syringae, while overexpression of ZIN1 results in enhanced susceptibility. Functionally, ZIN1 exhibits topoisomerase-like activity in vitro. Transcriptional profiling of wild-type and zin1 Arabidopsis plants inoculated with P. syringae indicated that while ZIN1 regulates a wide range of pathogen-responsive biological processes, the list of genes more highly expressed in zin1 versus wild-type plants was particularly enriched for ribosomal protein genes. Altogether, these data illuminate ZIN1 as a potential susceptibility hub that interacts with multiple effectors to influence the outcome of plant-microbe interactions.

scholarly journals Mechanism of action of rigosertib does not involve tubulin binding

2019 ◽  
Author(s):  
Stacey J. Baker ◽  
Stephen C. Cosenza ◽  
Saikrishna Athuluri-Divakar ◽  
M.V. Ramana Reddy ◽  
Rodrigo Vasquez-Del Carpio ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Human Tumor ◽  
Binding Activity ◽  
Phase Iii ◽  
Clinical Grade ◽  
Wild Type ◽  
Binding Domains ◽  
Tubulin Binding

SUMMARYRigosertib is a novel benzyl styryl sulfone that inhibits the growth of a wide variety of human tumor cells in vitro and in vivo and is currently in Phase III clinical trials. We recently provided structural and biochemical evidence to show that rigosertib acts as a RAS-mimetic by binding to Ras Binding Domains (RBDs) of the RAF and PI3K family proteins and disrupts their binding to RAS. In a recent study, Jost et al (2017) attributed the mechanism of action of rigosertib to microtubule-binding. In these studies, rigosertib was obtained from a commercial vendor. We have been unable to replicate the reported results with clinical grade rigosertib, and hence compared the purity of clinical grade and commercially sourced rigosertib. We find that the commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical grade rigosertib, which is free of this impurity, does not exhibit tubulin binding activity. In vivo, cell lines that express mutant β-tubulin (TUBBL240F) were also reported to be resistant to the effects of rigosertib. However, our studies showed that both wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Morphologically, we find that rigosertib, at lethal concentrations, induced a senescence-like phenotype in the small percentage of both wild-type and TUBBL240F-expressing cells that survive in the presence of rigosertib. Our results suggest that TUBBL240F expressing cells are more prone to undergo senescence in the presence of rigosertib as well as BI2536, an unrelated ATP-competitive pan-PLK inhibitor. The appearance of these senescent cells could be incorrectly scored as resistant cells in flow cytometric assays using short term cultures.

scholarly journals SUN-564 ATG7 Overexpression Results in Reduction of Hepatocellular Lipid Content in Vitro

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Federica Tavaglione ◽  
Guido Baselli ◽  
Ester Ciociola ◽  
Umberto Vespasiani Gentilucci ◽  
Luca Valenti ◽  
...  
Keyword(s):  
Liver Disease ◽  
Lipid Content ◽  
Lipid Droplets ◽  
Low Frequency ◽  
Underlying Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Intracellular Lipid ◽  
Heparg Cells

Abstract Abstract: Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, paralleling the epidemic of obesity and type 2 diabetes. Despite the high prevalence of NAFLD, only a minority of patients progress to NASH and advanced fibrosis. The reasons for this inter-individual variability are not completely understood but can be partially accounted for by genetic risk factors (1). Although several common genetic variants associated with liver disease have been identified, there is still a proportion of NAFLD heritability that remains unknown. The rare rs143545741 C>T variant in the autophagy related 7 (ATG7) gene (P426L) has been associated with a higher risk of progressive NAFLD (2). Interestingly, ATG7 encodes a E1-like ubiquitin activating enzyme which is involved in hepatic lipophagy (3). We hypothesized that the unknown heritability of NAFLD might be partially explained by rare genetic variants, therefore not identified in the GWAS studies. Moreover, we assumed that loss-of-function variants in ATG7 might confer an increased susceptibility to NAFLD by reducing autophagic catabolism of lipid droplets in the liver. To examine the underlying mechanism of the low-frequency V471A variant and the rare T86I, L127I, Q170E, and P426L variants in ATG7, we performed in vitro experiments of HepaRG cells overexpressing the human V5-tagged ATG7. We observed a reduction in intracellular lipid content in HepaRG cells overexpressing the ATG7 wild type and the 86I mutant protein (p=0.029, n=4) but not the 127I, 170E, 426L, and 471A mutant proteins. Cells with the ATG7 127I, 170E, 426L, and 471A mutants had higher intracellular lipid content compared to cells overexpressing the wild type protein (p=0.029, n=4). Our data suggested that the low-frequency V471A variant and the rare L127I, Q170E, and P426L variants in ATG7 are loss-of-function, resulting in defective lipophagy, reduced hepatocellular lipid droplets turnover, and excessive lipid accumulation. More experiments are needed to clarify the underlying mechanism of the T86I variant. In conclusion, we highlighted a role for ATG7 in reducing hepatocellular lipid content. Furthermore, we provided evidence showing non-synonymous variants in ATG7 increase the risk of NAFLD and that these variants are loss-of-function. We speculate that ATG7 might be a new susceptibility risk genetic locus for liver disease development and progression. References: (1) Eslam et al. J Hepatol. 2018;68(2):268–279. (2) Baselli et al. The Liver Meeting 2018 - AASLD. Hepatology. October 2018. Volume 68, Issue S1. (3) Martinez-Lopez and Singh. Annu Rev Nutr. 2015;35:215–37.

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