scholarly journals Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia

2018 ◽  
Vol 19 (10) ◽  
pp. 3099 ◽  
Author(s):  
Anna Malekkou ◽  
Maura Samarani ◽  
Anthi Drousiotou ◽  
Christina Votsi ◽  
Sandro Sonnino ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Autosomal Recessive ◽  
Biochemical Characterization ◽  
Fold Increase ◽  
Loss Of Function ◽  
Spastic Paraplegia ◽  
Spastic Ataxia ◽  
Autosomal Recessive Cerebellar Ataxia ◽  
Compensatory Effect

The GBA2 gene encodes the non-lysosomal glucosylceramidase (NLGase), an enzyme that catalyzes the conversion of glucosylceramide (GlcCer) to ceramide and glucose. Mutations in GBA2 have been associated with the development of neurological disorders such as autosomal recessive cerebellar ataxia, hereditary spastic paraplegia, and Marinesco-Sjogren-Like Syndrome. Our group has previously identified the GBA2 c.1780G>C [p.Asp594His] missense mutation, in a Cypriot consanguineous family with spastic ataxia. In this study, we carried out a biochemical characterization of lymphoblastoid cell lines (LCLs) derived from three patients of this family. We found that the mutation strongly reduce NLGase activity both intracellularly and at the plasma membrane level. Additionally, we observed a two-fold increase of GlcCer content in LCLs derived from patients compared to controls, with the C16 lipid being the most abundant GlcCer species. Moreover, we showed that there is an apparent compensatory effect between NLGase and the lysosomal glucosylceramidase (GCase), since we found that the activity of GCase was three-fold higher in LCLs derived from patients compared to controls. We conclude that the c.1780G>C mutation results in NLGase loss of function with abolishment of the enzymatic activity and accumulation of GlcCer accompanied by a compensatory increase in GCase.

Characterization of a novel loss-of-function variant in TDP2 in two adult patients with spinocerebellar ataxia autosomal recessive 23 (SCAR23)

2020 ◽  
Vol 65 (12) ◽  
pp. 1135-1141
Author(s):  
Edoardo Errichiello ◽  
Guido Zagnoli-Vieira ◽  
Romana Rizzi ◽  
Livia Garavelli ◽  
Keith W. Caldecott ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia ◽  
Autosomal Recessive ◽  
Adult Patients ◽  
Loss Of Function

scholarly journals Characterization of a novel loss-of-function variant in TDP2 in two adult patients with spinocerebellar ataxia autosomal recessive 23 (SCAR23)

Authorea ◽  
2020 ◽  
Author(s):  
Edoardo Errichiello ◽  
Guido Zagnoli Vieira ◽  
Romana Rizzi ◽  
Livia Garavelli ◽  
Keith Caldecott ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia ◽  
Autosomal Recessive ◽  
Adult Patients ◽  
Loss Of Function

scholarly journals The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes

2018 ◽  
Vol 29 (8) ◽  
pp. 2110-2122 ◽  
Author(s):  
Gentzon Hall ◽  
Brandon M. Lane ◽  
Kamal Khan ◽  
Igor Pediaditakis ◽  
Jianqiu Xiao ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Adaptor Protein ◽  
Pi3k Pathway ◽  
Actin Binding ◽  
Loss Of Function ◽  
Signaling Axis ◽  
Phosphoinositide 3 Kinase

BackgroundWe previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.MethodsWe conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT) or ANLNR431C.ResultsExperiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.ConclusionsThe ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

scholarly journals GDAP2 mutations implicate susceptibility to cellular stress in a new form of cerebellar ataxia

Brain ◽  
2018 ◽  
Vol 141 (9) ◽  
pp. 2592-2604 ◽  
Author(s):  
Ilse Eidhof ◽  
Jonathan Baets ◽  
Erik-Jan Kamsteeg ◽  
Tine Deconinck ◽  
Lisa van Ninhuijs ◽  
...  
Keyword(s):  
Cerebellar Ataxia ◽  
Autosomal Recessive ◽  
Cellular Stress ◽  
Orthologous Gene ◽  
Causative Role ◽  
Loss Of Function ◽  
Autosomal Recessive Cerebellar Ataxia ◽  
Pyramidal Tracts ◽  
Cerebellar Ataxias ◽  
Increased Sensitivity

Abstract Autosomal recessive cerebellar ataxias are a group of rare disorders that share progressive degeneration of the cerebellum and associated tracts as the main hallmark. Here, we report two unrelated patients with a new subtype of autosomal recessive cerebellar ataxia caused by biallelic, gene-disruptive mutations inGDAP2, a gene previously not implicated in disease. Both patients had onset of ataxia in the fourth decade. Other features included progressive spasticity and dementia. Neuropathological examination showed degenerative changes in the cerebellum, olive inferior, thalamus, substantia nigra, and pyramidal tracts, as well as tau pathology in the hippocampus and amygdala. To provide further evidence for a causative role ofGDAP2 mutations in autosomal recessive cerebellar ataxia pathophysiology, its orthologous gene was investigated in the fruit flyDrosophila melanogaster. Ubiquitous knockdown ofDrosophila Gdap2 resulted in shortened lifespan and motor behaviour anomalies such as righting defects, reduced and uncoordinated walking behaviour, and compromised flight. Gdap2 expression levels responded to stress treatments in control flies, and Gdap2 knockdown flies showed increased sensitivity to deleterious effects of stressors such as reactive oxygen species and nutrient deprivation. Thus,Gdap2 knockdown inDrosophila andGDAP2 loss-of-function mutations in humans lead to locomotor phenotypes, which may be mediated by altered responses to cellular stress.

scholarly journals Expression and biochemical characterization of human glucose-6- phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1436-1441 ◽  
Author(s):  
TK Tang ◽  
CH Yeh ◽  
CS Huang ◽  
MJ Huang
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Fold Increase ◽  
Binding Domain ◽  
Occurrence Rate ◽  
Substrate Analogues ◽  
Normal Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase

We have developed a system to characterize normal and mutated glucose-6- phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->wG; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.

scholarly journals Expression and biochemical characterization of human glucose-6- phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1436-1441 ◽  
Author(s):  
TK Tang ◽  
CH Yeh ◽  
CS Huang ◽  
MJ Huang
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Fold Increase ◽  
Binding Domain ◽  
Occurrence Rate ◽  
Substrate Analogues ◽  
Normal Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract We have developed a system to characterize normal and mutated glucose-6- phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->wG; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.

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