A Drosophila Mitochondrial Complex I Deficiency Phenotype Array

Mitochondrial diseases are a group of rare life-threatening diseases often caused by defects in the oxidative phosphorylation system. No effective treatment is available for these disorders. Therapeutic development is hampered by the high heterogeneity in genetic, biochemical, and clinical spectra of mitochondrial diseases and by limited preclinical resources to screen and identify effective treatment candidates. Alternative models of the pathology are essential to better understand mitochondrial diseases and to accelerate the development of new therapeutics. The fruit fly Drosophila melanogaster is a cost- and time-efficient model that can recapitulate a wide range of phenotypes observed in patients suffering from mitochondrial disorders. We targeted three important subunits of complex I of the mitochondrial oxidative phosphorylation system with the flexible UAS-Gal4 system and RNA interference (RNAi): NDUFS4 (ND-18), NDUFS7 (ND-20), and NDUFV1 (ND-51). Using two ubiquitous driver lines at two temperatures, we established a collection of phenotypes relevant to complex I deficiencies. Our data offer models and phenotypes with different levels of severity that can be used for future therapeutic screenings. These include qualitative phenotypes that are amenable to high-throughput drug screening and quantitative phenotypes that require more resources but are likely to have increased potential and sensitivity to show modulation by drug treatment

Mutations in SELENBP1, encoding a novel human methanethiol oxidase, cause extraoral halitosis

Selenium-binding protein 1 (SELENBP1) has been associated with several cancers, although its exact role is unknown. We show that SELENBP1 is a methanethiol oxidase (MTO), related to the MTO in methylotrophic bacteria, that converts methanethiol to H2O2, formaldehyde, and H2S, an activity not previously known to exist in humans. We identified mutations in SELENBP1 in five patients with cabbage-like breath odor. The malodor was attributable to high levels of methanethiol and dimethylsulfide, the main odorous compounds in their breath. Elevated urinary excretion of dimethylsulfoxide was associated with MTO deficiency. Patient fibroblasts had low SELENBP1 protein levels and were deficient in MTO enzymatic activity; these effects were reversed by lentivirus-mediated expression of wild-type SELENBP1. Selenbp1-knockout mice showed biochemical characteristics similar to those in humans. Our data reveal a potentially frequent inborn error of metabolism that results from MTO deficiency and leads to a malodor syndrome

Mutations in SELENBP1, encoding a novel human methanethiol oxidase, cause extraoral halitosis

Selenium-binding protein 1 (SELENBP1) has been associated with several cancers, although its exact role is unknown. We show that SELENBP1 is a methanethiol oxidase (MTO), related to the MTO in methylotrophic bacteria, that converts methanethiol to H2O2, formaldehyde, and H2S, an activity not previously known to exist in humans. We identified mutations in SELENBP1 in five patients with cabbage-like breath odor. The malodor was attributable to high levels of methanethiol and dimethylsulfide, the main odorous compounds in their breath. Elevated urinary excretion of dimethylsulfoxide was associated with MTO deficiency. Patient fibroblasts had low SELENBP1 protein levels and were deficient in MTO enzymatic activity; these effects were reversed by lentivirus-mediated expression of wild-type SELENBP1. Selenbp1-knockout mice showed biochemical characteristics similar to those in humans. Our data reveal a potentially frequent inborn error of metabolism that results from MTO deficiency and leads to a malodor syndrome.info:eu-repo/semantics/publishedVersio

Nef Associates with p21-Activated Kinase 2 in a p21-GTPase-Dependent Dynamic Activation Complex within Lipid Rafts

We have previously reported that Nef specifically interacts with a small but highly active subpopulation of p21-activated kinase 2 (PAK2). Here we show that this is due to a transient association of Nef with a PAK2 activation complex within a detergent-insoluble membrane compartment containing the lipid raft marker GM1. The low abundance of this Nef-associated kinase (NAK) complex was found to be due to an autoregulatory mechanism. Although activation of PAK2 was required for assembly of the NAK complex, catalytic activity of PAK2 also promoted dissociation of this complex. Testing different constitutively active PAK2 mutants indicated that the conformation associated with p21-mediated activation rather than kinase activity per se was required for PAK2 to become NAK. Although association with PAK2 is one of the most conserved properties of Nef, we found that the ability to stimulate PAK2 activity differed markedly among divergent Nef alleles, suggesting that PAK2 association and activation are distinct functions of Nef. However, mutations introduced into the p21-binding domain of PAK2 revealed that p21-GTPases are involved in both of these Nef functions and, in addition to promoting PAK2 activation, also help to physically stabilize the NAK complex

The biology of the Gaucher cell: the cradle of human chitinases

Gaucher disease (GD) is the most common lysosomal storage disorder and is caused by inherited deficiencies of glucocerebrosidase, the enzyme responsible for the lysosomal breakdown of the lipid glucosylceramide. GD is characterized by the accumulation of pathological, lipid laden macrophages, so-called Gaucher cells. Following the development of enzyme replacement therapy for GD, the search for suitable surrogate disease markers resulted in the identification of a thousand-fold increased chitinase activity in plasma from symptomatic Gaucher patients and that decreases upon successful therapeutic intervention. Biochemical investigations identified a single enzyme, named chitotriosidase, to be responsible for this activity. Chitotriosidase was found to be an excellent marker for lipid laden macrophages in Gaucher patients and is now widely used to assist clinical management of patients. In the wake of the identification of chitotriosidase, the presence of other members of the chitinase family in mammals was discovered. Amongst these is AMCase, an enzyme recently implicated in the pathogenesis of asthma. Chitinases are omnipresent throughout nature and are also produced by vertebrates in which they play important roles in defence against chitin-containing pathogens and in food processin

CCL18: a urinary marker of Gaucher cell burden in Gaucher patients

Glucosylceramide-laden tissue macrophages in Gaucher patients secrete large quantities of chitotriosidase and CC chemokine ligand 18 (CCL18), resulting in markedly increased plasma levels. We have comparatively investigated the occurrence of both parameters in plasma and urine samples of Gaucher patients. Chitotriosidase was high in urine samples of some symptomatic patients, but elevations did not correlate with increased plasma concentrations. Urinary chitotriosidase was particularly high in a patient with severe kidney involvement and local storage cell infiltration. Urinary levels of CCL18 were also highly elevated in samples from Gaucher patients as compared to controls. The median value of the CCL18/creatinine ratio in urine samples of 31 Gaucher patients was 143.3 pg/micromol (range 32-551) and in those of 12 normal subjects was 4.1 pg/micromol (range 1.3-6.8). In sharp contrast to chitotriosidase, increases in the low-molecular-mass chemokine CCL18 in urine and plasma specimens of Gaucher patients correlated well. A correlation was also observed for reductions in urinary and plasma CCL18 following therapy. It is concluded that assessment of urinary CCL18 of Gaucher patients gives insight into the total body burden on Gaucher cells, whereas that of chitotriosidase does not. Urinary chitotriosidase appears rather to be a reflection of renal patholog