scholarly journals Functional characterization of a novel loss-of-function mutation of PRPS1 related to early-onset progressive nonsyndromic hearing loss in Koreans (DFNX1): Potential implications on future therapeutic intervention

2016 ◽  
Vol 18 (11-12) ◽  
pp. 353-358 ◽  
Author(s):  
So Young Kim ◽  
Ah Reum Kim ◽  
Nayoung K.D. Kim ◽  
Chung Lee ◽  
Jin Hee Han ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Therapeutic Intervention ◽  
Early Onset ◽  
Functional Characterization ◽  
Nonsyndromic Hearing Loss ◽  
Loss Of Function

scholarly journals Functional Characterization of the Adenylyl Cyclase Genesgs-1by Analysis of a Mutational Spectrum inCaenorhabditis elegans

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 133-142 ◽  
Author(s):  
Celine Moorman ◽  
Ronald H A Plasterk
Keyword(s):  
Life Span ◽  
Adenylyl Cyclase ◽  
Neuronal Degeneration ◽  
Functional Characterization ◽  
Adenylyl Cyclases ◽  
Loss Of Function ◽  
C Elegans ◽  
Larval Stages ◽  
Pharyngeal Pumping

AbstractThe sgs-1 (suppressor of activated Gαs) gene encodes one of the four adenylyl cyclases in the nematode C. elegans and is most similar to mammalian adenylyl cyclase type IX. We isolated a complete loss-of-function mutation in sgs-1 and found it to result in animals with retarded development that arrest in variable larval stages. sgs-1 mutant animals exhibit lethargic movement and pharyngeal pumping and (while not reaching adulthood) have a mean life span that is >50% extended compared to wild type. An extensive set of reduction-of-function mutations in sgs-1 was isolated in a screen for suppressors of a neuronal degeneration phenotype induced by the expression of a constitutively active version of the heterotrimeric Gαs subunit of C. elegans. Although most of these mutations change conserved residues within the catalytic domains of sgs-1, mutations in the less-conserved transmembrane domains are also found. The sgs-1 reduction-of-function mutants are viable and have reduced locomotion rates, but do not show defects in pharyngeal pumping or life span.

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

scholarly journals Molecular and functional characterization of detrusor PDGFRα positive cells in spinal cord injury-induced detrusor overactivity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ken Lee ◽  
Sang O Park ◽  
Pil-Cho Choi ◽  
Seung-Bum Ryoo ◽  
Haeyeong Lee ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Detrusor Overactivity ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Interstitial Cells ◽  
Sk Channels ◽  
Loss Of Function ◽  
Cord Injury

AbstractVolume accommodation occurs via a novel mechanism involving interstitial cells in detrusor muscles. The interstitial cells in the bladder are PDGFRα+, and they restrain the excitability of smooth muscle at low levels and prevents the development of transient contractions (TCs). A common clinical manifestation of spinal cord injury (SCI)-induced bladder dysfunction is detrusor overactivity (DO). Although a myogenic origin of DO after SCI has been suggested, a mechanism for development of SCI-induced DO has not been determined. In this study we hypothesized that SCI-induced DO is related to loss of function in the regulatory mechanism provided by PDGFRα+ cells. Our results showed that transcriptional expression of Pdgfra and Kcnn3 was decreased after SCI. Proteins encoded by these genes also decreased after SCI, and a reduction in PDGFRα+ cell density was also documented. Loss of PDGFRα+ cells was due to apoptosis. TCs in ex vivo bladders during filling increased dramatically after SCI, and this was related to the loss of regulation provided by SK channels, as we observed decreased sensitivity to apamin. These findings show that damage to the mechanism restraining muscle contraction during bladder filling that is provided by PDGFRα+ cells is causative in the development of DO after SCI.

A Novel Loss of Function Melanocortin-4-Receptor Mutation (MC4R-F313Sfs*29) in Morbid Obesity

2020 ◽  
Author(s):  
Elisabetta Trevellin ◽  
Marnie Granzotto ◽  
Cristina Host ◽  
Francesca Grisan ◽  
Diego De Stefani ◽  
...  
Keyword(s):  
Early Onset ◽  
Functional Characterization ◽  
Gene Mutations ◽  
Melanocortin Receptor ◽  
Hek293 Cells ◽  
Intracellular Camp ◽  
Loss Of Function ◽  
Mc4r Gene ◽  
Melanocortin 4 Receptor

Abstract Context Melanocortin receptor-4 (MC4R) gene mutations are associated with early-onset severe obesity, and the identification of potential pathological variants is crucial for the clinical management of patients with obesity. Objective To explore whether and how a novel heterozygous MC4R variant (MC4R-F313Sfs*29), identified in a young boy (body mass index [BMI] 38.8 kg/m2) during a mutation analysis conducted in a cohort of patients with obesity, plays a determinant pathophysiological role in the obesity development. Design Setting and Patients The genetic screening was carried out in a total of 209 unrelated patients with obesity (BMI ≥ 35 kg/m2). Structural and functional characterization of the F313Sfs*29-mutated MC4R was performed using computational approaches and in vitro, using HEK293 cells transfected with genetically encoded biosensors for cAMP and Ca2+. Results The F313Sfs*29 was the only variant identified. In vitro experiments showed that HEK293 cells transfected with the mutated form of MC4R did not increase intracellular cAMP or Ca2+ levels after stimulation with a specific agonist in comparison with HEK293 cells transfected with the wild type form of MC4R (∆R/R0 = -90% ± 8%; P < 0.001). In silico modeling showed that the F313Sfs*29 mutation causes a major reorganization in the cytosolic domain of MC4R, thus reducing the affinity of the putative GalphaS binding site. Conclusions The newly discovered F313Sfs*29 variant of MC4R may be involved in the impairment of α-MSH-induced cAMP and Ca2+ signaling, blunting intracellular G protein-mediated signal transduction. This alteration might have led to the dysregulation of satiety signaling, resulting in hyperphagia and early onset of obesity.

Functional characterization of a new human melanocortin-4 receptor homozygous mutation (N72K) that is associated with early-onset obesity

2014 ◽  
Vol 41 (12) ◽  
pp. 7967-7972 ◽  
Author(s):  
Patric J. D. Delhanty ◽  
Elise Bouw ◽  
Martin Huisman ◽  
Resie M. L. Vervenne ◽  
Axel P. N. Themmen ◽  
...  
Keyword(s):  
Early Onset ◽  
Functional Characterization ◽  
Homozygous Mutation ◽  
Melanocortin 4 Receptor

scholarly journals Novel Loss-of-Function Mutations in COCH Cause Autosomal Recessive Nonsyndromic Deafness

2020 ◽  
Author(s):  
Kevin T Booth ◽  
Amama Ghaffar ◽  
Muhammad Rashid ◽  
Luke T Hovey ◽  
Mureed Hussain ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Rna Splicing ◽  
Autosomal Recessive ◽  
Dominant Negative ◽  
Nonsyndromic Hearing Loss ◽  
Loss Of Function ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Exon Splicing ◽  
Coding Variants

AbstractCOCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly deafness in mice and humans. Two forms of deafness are linked to mutations in COCH, the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used mini-gene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA-splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA-splicing, and highlights the need to investigate the effect of coding variants on RNA-splicing.

scholarly journals Structural and functional characterization of the mitochondrial complex IV assembly factor Coa6

2019 ◽  
Vol 2 (5) ◽  
pp. e201900458 ◽  
Author(s):  
Shadi Maghool ◽  
N Dinesha G Cooray ◽  
David A Stroud ◽  
David Aragão ◽  
Michael T Ryan ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Protein Complexes ◽  
Functional Characterization ◽  
Copper Binding ◽  
Loss Of Function ◽  
Structural Explanation ◽  
Complex Iv ◽  
Assembly Factor ◽  
Bundle Structure

Assembly factors play key roles in the biogenesis of many multi-subunit protein complexes regulating their stability, activity, and the incorporation of essential cofactors. The human assembly factor Coa6 participates in the biogenesis of the CuA site in complex IV (cytochrome c oxidase, COX). Patients with mutations in Coa6 suffer from mitochondrial disease due to complex IV deficiency. Here, we present the crystal structures of human Coa6 and the pathogenic W59CCoa6-mutant protein. These structures show that Coa6 has a 3-helical bundle structure, with the first 2 helices tethered by disulfide bonds, one of which likely provides the copper-binding site. Disulfide-mediated oligomerization of the W59CCoa6 protein provides a structural explanation for the loss-of-function mutation.

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