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3.
Biosci Rep ; 37(2)2017 04 30.
Article in English | MEDLINE | ID: mdl-28143957

ABSTRACT

In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, ß, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85-p110 interactions typical of cancer.


Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/genetics , Mutation , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Growth Disorders/enzymology , Growth Disorders/genetics , Growth Disorders/pathology , Humans , Hypercalcemia/enzymology , Hypercalcemia/genetics , Hypercalcemia/pathology , Immunoblotting , Metabolic Diseases/enzymology , Metabolic Diseases/genetics , Metabolic Diseases/pathology , Models, Biological , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/pathology , Nephrocalcinosis/enzymology , Nephrocalcinosis/genetics , Nephrocalcinosis/pathology , Protein Subunits/genetics , Protein Subunits/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
4.
J Inherit Metab Dis ; 38(2): 211-9, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25595726

ABSTRACT

Whole exome sequencing was used to investigate the genetic cause of mitochondrial disease in two siblings with a syndrome of congenital lamellar cataracts associated with nephrocalcinosis, medullary cysts and 3-methylglutaconic aciduria. Autosomal recessive inheritance in a gene encoding a mitochondrially targeted protein was assumed; the only variants which satisfied these criteria were c.1882C>T (p.Arg628Cys) and c.1915G>A (p.Glu639Lys) in the CLPB gene, encoding a heat shock protein/chaperonin responsible for disaggregating mitochondrial and cytosolic proteins. Functional studies, including quantitative PCR (qPCR) and Western blot, support pathogenicity of these mutations. Furthermore, molecular modelling suggests that the mutations disrupt interactions between subunits so that the CLPB hexamer cannot form or is unstable, thus impairing its role as a protein disaggregase. We conclude that accumulation of protein aggregates underlies the development of cataracts and nephrocalcinosis in CLPB deficiency, which is a novel genetic cause of 3-methylglutaconic aciduria. A common mitochondrial cause for 3-methylglutaconic aciduria appears to be disruption of the architecture of the mitochondrial membranes, as in Barth syndrome (tafazzin deficiency), Sengers syndrome (acylglycerol kinase deficiency) and MEGDEL syndrome (impaired remodelling of the mitochondrial membrane lipids because of SERAC1 mutations). We now propose that perturbation of the mitochondrial membranes by abnormal protein aggregates leads to 3-methylglutaconic aciduria in CLPB deficiency.


Subject(s)
Cataract/genetics , Endopeptidase Clp/genetics , Kidney Diseases, Cystic/genetics , Metabolism, Inborn Errors/genetics , Mitochondrial Diseases/genetics , Mutation , Nephrocalcinosis/genetics , Cataract/diagnosis , Cataract/enzymology , Cells, Cultured , DNA Mutational Analysis , Endopeptidase Clp/chemistry , Endopeptidase Clp/deficiency , Exome , Female , Genetic Predisposition to Disease , Heredity , Humans , Kidney Diseases, Cystic/diagnosis , Kidney Diseases, Cystic/enzymology , Male , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/enzymology , Mitochondrial Diseases/diagnosis , Mitochondrial Diseases/enzymology , Mitochondrial Membranes/pathology , Models, Molecular , Nephrocalcinosis/diagnosis , Nephrocalcinosis/enzymology , Pedigree , Phenotype , Protein Aggregation, Pathological , Protein Conformation , Risk Factors , Siblings , Structure-Activity Relationship
5.
Fish Shellfish Immunol ; 42(1): 184-92, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25449383

ABSTRACT

Infectious diseases and breeding conditions can influence fish health status. Furthermore it is well known that human and animal health are strongly correlated. In lower vertebrates melano-macrophage centres, clusters of pigment-containing cells forming the extracutaneous pigment system, are widespread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. Hence, they are employed as biomarker of fish health status. We have investigated this cell system in the European sea bass (Dicentrarchus labrax L.) following the enzyme activities involved in melanin biosynthesis. We have found a possible relationship between kidney disease of farmed fishes and dopa oxidase activity level, suggesting it as an indicator of kidney disease. Moreover variations of dopa oxidase activity in extracutaneous pigment system have been observed with respect to environmental temperature. At last, for the first time, using femtosecond transient absorption spectroscopy (Femto-TA), we pointed out that pigment-containing cells of fish kidney tissue present melanin pigments.


Subject(s)
Bass , Biomarkers/metabolism , Fish Diseases/enzymology , Monophenol Monooxygenase/metabolism , Nephrocalcinosis/veterinary , Pigments, Biological/metabolism , Animals , Aquaculture , Electrophoresis, Polyacrylamide Gel , Melanins/biosynthesis , Nephrocalcinosis/enzymology , Peroxidase/metabolism , Tyrosine 3-Monooxygenase/metabolism , X-Ray Absorption Spectroscopy
7.
Am J Hum Genet ; 93(1): 150-7, 2013 Jul 11.
Article in English | MEDLINE | ID: mdl-23810379

ABSTRACT

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.


Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/metabolism , Growth Disorders/enzymology , Hypercalcemia/enzymology , Metabolic Diseases/enzymology , Nephrocalcinosis/enzymology , Signal Transduction , Adipocytes/metabolism , Adolescent , Adult , Aged , Amino Acid Sequence , Body Mass Index , Cell Differentiation , Class Ia Phosphatidylinositol 3-Kinase/genetics , DNA Mutational Analysis , Enzyme Activation , Exome , Female , Fibroblasts/metabolism , Gene Knockout Techniques , Genetic Carrier Screening , Genetic Predisposition to Disease , Genetics, Population/methods , Growth Disorders/pathology , Humans , Hypercalcemia/pathology , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Male , Metabolic Diseases/pathology , Middle Aged , Molecular Sequence Data , Mutation , Nephrocalcinosis/pathology , Pedigree , Young Adult , src Homology Domains
8.
J Inherit Metab Dis ; 33 Suppl 3: S21-4, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20077140

ABSTRACT

UNLABELLED: This report describes the clinical, biochemical and molecular data of a 78-year-old patient with xanthine dehydrogenase deficiency presenting as rheumatoid arthritis. BACKGROUND: Xanthinuria type I is a rare disorder of purine metabolism caused by xanthine dehydrogenase (XDH) deficiency; fewer than 150 cases have been described in the literature so far. METHODS: We describe the clinical history and urine and serum findings of a 78-year-old patient with isolated XDH deficiency presenting as rheumatoid arthritis. The diagnosis was confirmed by mutation analysis. RESULTS: The patient suffered from arthral symptoms and nephrocalcinosis. Very low concentrations of uric acid were observed in her serum and urine. The allopurinol loading test indicated her xanthinuria to be type I. Analysis of genomic DNA revealed novel heterozygous deletion in exon 8 (g.27073delC, p.214QfsX4) and previously published heterozygous nucleotide missense transition in exon 25 (g.64772-C>T, p.T910M). CONCLUSION: Hereditary xanthinuria is a rare disorder, but it also needs to be considered in patients not originating from Mediterranean countries or the Near or Middle East. Urate concentration in serum and urine may provide an initial indication of XDH deficiency before high-performance liquid chromatography (HPLC) analysis is performed. The key to identifying the disorder is a greater awareness of XDH deficiency amongst primary care physicians, nephrologists, and urologists, but also rheumatologists. The diagnosis and therapeutic management requires a multidisciplinary approach.


Subject(s)
Arthritis, Rheumatoid/genetics , Metabolism, Inborn Errors/genetics , Mutation, Missense , Sequence Deletion , Xanthine Dehydrogenase/deficiency , Xanthine Dehydrogenase/genetics , Aged , Allopurinol , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/enzymology , Biomarkers/blood , Biomarkers/urine , Chromatography, High Pressure Liquid , DNA Mutational Analysis , Exons , Female , Genetic Predisposition to Disease , Genetic Testing/methods , Heterozygote , Humans , Metabolism, Inborn Errors/complications , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/enzymology , Nephrocalcinosis/enzymology , Nephrocalcinosis/genetics , Phenotype , Predictive Value of Tests , Uric Acid/blood , Uric Acid/urine
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