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The enzyme kynurenine 3-monooxygenase (KMO) operates at a critical branch-point in the kynurenine pathway (KP), the major route of tryptophan metabolism. As the KP has been implicated in the pathogenesis of several human diseases, KMO and other enzymes that control metabolic flux through the pathway are potential therapeutic targets for these disorders. While KMO is localized to the outer mitochondrial membrane in eukaryotic organisms, no mitochondrial role for KMO has been described. In this study, KMO deficient Drosophila melanogaster were investigated for mitochondrial phenotypes in vitro and in vivo. We find that a loss of function allele or RNAi knockdown of the Drosophila KMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably, cinnabar genetically interacts with the Parkinson’s disease associated genes Pink1 and parkin, as well as the mitochondrial fission gene Drp1, implicating KMO in mitochondrial dynamics and mitophagy, mechanisms which govern the maintenance of a healthy mitochondrial network. Overexpression of human KMO in mammalian cells finds that KMO plays a role in the post-translational regulation of DRP1. These findings reveal a novel mitochondrial role for KMO, independent from its enzymatic role in the kynurenine pathway., Author summary Mitochondria are subject to quality control mechanisms to maintain homeostasis, regularly undergoing fission and fusion to rescue damaged organelles or segregate regions that are damaged beyond repair which are then cleared by mitophagy. These quality control mechanisms have been implicated in a number of neurodegenerative diseases, including familial Parkinson’s disease, which can be caused by mutations in two mitophagy governing genes, PINK1 and PRKN. The kynurenine pathway is a pathway through which dietary tryptophan is metabolised. The pathway has multiple branchpoints and an imbalance in flux through these branches has been associated with neurodegenerative disease. The enzyme kynurenine 3-monooxygenase (KMO) sits at a critical branchpoint controlling balance in the pathway. KMO is localised to mitochondria, yet to date any mitochondria-specific function is unknown. In this study, we demonstrate that KMO plays a role in mitochondrial fission via the fission factor DRP1 and that KMO deficiency leads to changes in mitochondrial morphology and function. Manipulation of KMO in Drosophila melanogaster also demonstrates an interaction with PINK1 and PRKN. Intriguingly, these interactions appear to be independent of the known enzymatic role of KMO in the kynurenine pathway, demonstrating a novel and independent function of the protein.

Background Metabolic dysfunction is a hallmark of HD pathology. Decreased glucose metabolism and increased lactate concentrations in HD brains suggest mitochondrial involvement in the disease. Moreover, alterations in mitochondrial fission/fusion balance have also been reported in several HD models, with repercussions on the ability of cells to clear damaged mitochondria. Aims Here we employ a Drosophila model of HD to study mitochondrial and neuronal dysfunction. We investigate the overexpression of Parkin, an E3 ubiquitin ligase involved in mitochondrial fission/fusion and quality control, as well as the expression of an alternative oxidase (AOX) which bypasses the activity of the respiratory complexes III and IV. Methods High resolution respirometry was performed with an Oroboros O2K Oxygraph which permitted measurement of mitochondrial leak, oxidative phosphorylation (oxphos) capacity, electron transfer system capacity and cytochrome C loss. Mitochondrial shape and mass were assessed by electron microscopy and citrate synthase activity. Neuronal phenotypes were studied by measuring the loss of eye rhabdomeres, deficits in eclosion from the pupal case and the longevity of adult flies. Results Overexpression of Parkin in HD muscles significantly improved mitochondrial function by reducing mitochondrial leak, increasing the oxphos and electron transfer system capacity of Complex I and I+II, and reducing cytochrome C loss. Moreover Parkin overexpression increased mitochondrial fission and autophagy. All neuronally associated defects were robustly rescued by coexpression of Parkin in HD flies. Bypassing mitochondrial defects of complex III and IV via AOX also resulted in amelioration of all neuronal phenotypes and provided a striking rescue of shortened lifespan in HD flies. Conclusions This study highlights the likely contribution of mitochondrial dysfunction to HD and the importance of improving mitochondrial function and quality as a potential therapeutic intervention.

Banushi, Blerida/0000-0002-4314-8369; Dionisi-Vici, Carlo/0000-0002-0007-3379; Gissen, Paul/0000-0002-9712-6122; Coward, Richard/0000-0001-6183-2546; Kim, Chong/0000-0002-1754-1300; Galmes, Romain/0000-0001-5616-5937 WOS: 000310975900007 PubMed: 22753090 Arthrogryposisrenal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apicalbasolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotypephenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR. Hum Mutat 33:16561664, 2012. (c) 2012 Wiley Periodicals, Inc. Wellcome TrustWellcome Trust [WT095662MA]; Bold FP7 ITN project [238821]; VICI of the Netherlands Organization for Scientific Research [918.56.611]; British Heart FoundationBritish Heart Foundation [RG/09/007/27917]; Kidney Research UKKidney Research UK (KRUK) [RP22/2012]; Medical Research CouncilMedical Research Council UK (MRC) [G0501901, G9818340B]; Great Ormond Street Hospital Childrens Charity [ICH1034] Contract grant sponsors: H.S. is an MRC PhD fellow; P.G. is a Wellcome Trust Senior Research Fellow in Clinical Sciences (WT095662MA); P.G. and B.B. are supported by Bold FP7 ITN project-238821; R.G. and J.K. were supported by VICI grant 918.56.611 of the Netherlands Organization for Scientific Research awarded to J.K.

Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues., Lysine hydroxylation of procollagen precursors by LH3 is required for collagen fibril crosslinking and stabilization. Here the authors show that the trafficking protein VIPAR is required for correct sorting of LH3 into post-Golgi collagen carriers and for correct collagen modification and structure.

Background. Arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome is a multisystem autosomal-recessive disorder caused by defects in the VPS33B and VIPAR genes, involved in localization of apical membrane proteins. Affected children usually die by 1 year of age, often secondary to infective complications. The classic renal manifestation previously described in ARC syndrome is proximal–tubular dysfunction. The aim of this study is to gain further insight into the renal manifestations of this syndrome. Methods. Clinical review of three cases of ARC syndrome presenting to a tertiary centre. Together with measurement of VPS33B and VIPAR protein expression in the human glomerulus. Results. The cases demonstrated severe failure to thrive and in addition to commonly described features profound proteinuria and albuminuria, together with hypoalbuminaemia, suggesting glomerular involvement of this syndrome. Western blotting of conditionally immortalized human glomerular cells and ex vivo immunofluorescent analysis of the human glomerulus revealed that VPS33B and VIPAR were highly expressed in glomerular endothelium, and podocytes, but not in the mesangium. Conclusions. ARC syndrome affects the glomerulus as well as the proximal tubule in the kidney. Our molecular studies suggest that both cell types that constitute the glomerular filtration barrier are affected in this condition, providing an explanation for the albuminuria that we have observed in our cases.

Inherited immunodeficiency disorders can be caused by mutations in any one of a large number of genes involved in the function of immune cells. Here, we describe two families with an autosomal recessive inherited immunodeficiency disorder characterized by increased susceptibility to infection and autoimmunity. Genetic linkage studies mapped the disorder to chromosomal region 14q11.2, and a homozygous guanine-to-adenine substitution was identified at the last base of exon 3 immediately following the translational termination codon in the TCRα subunit constant gene (TRAC). RT-PCR analysis in the two affected individuals revealed impaired splicing of the mRNA, as exon 3 was lost from the TRAC transcript. The mutant TCRα chain protein was predicted to lack part of the connecting peptide domain and all of the transmembrane and cytoplasmic domains, which have a critical role in the regulation of the assembly and/or intracellular transport of TCR complexes. We found that T cells from affected individuals were profoundly impaired for surface expression of the TCRαβ complex. We believe this to be the first report of a disease-causing human TRAC mutation. Although the absence of TCRαβ+ T cells in the affected individuals was associated with immune dysregulation and autoimmunity, they had a surprising level of protection against infection.

Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar-and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney. Children Liver Disease Foundation Framework 6 IP EUTRACC (LSGH CT 2006037445) European Molecular Biology Organization (EMBO) (ASTF 121:2007) European Science Foundation (ESF) European Commission (2008) UK Research & Innovation (UKRI) Biotechnology and Biological Sciences Research Council (BBSRC) (BB/H002308/1) ARC syndrome association Children Living with Inherited Metabolic Diseases (CLIMB) Birmingham Children's Hospital Research Foundation (BCHRF) WellChild Wellcome Trust European Commission United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) (ZIAHD008807) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) (R01DK035652) NIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) (P30DK034928)

Arthrogryposis, Renal dysfunction and Cholestasis (ARC) syndrome is a multi-system autosomal recessive disorder caused by germline mutations in VPS33B. The detection of germline VPS33B mutations removes the need for diagnostic organ biopsies (these carry a >50% risk of life-threatening haemorrhage due to platelet dysfunction); however, VPS33B mutations are not detectable in similar to 25% of patients. In order further to define the molecular basis of ARC we performed mutation analysis and mRNA and protein studies in patients with a clinical diagnosis of ARC. Here we report novel mutations in VPS33B in patients from Eastern Europe and South East Asia. One of the mutations was present in 7 unrelated Korean patients. Reduced expression of VPS33B and cellular phenotype was detected in fibroblasts from patients clinically diagnosed with ARC with and without known VPS33B mutations. One mutation-negative patient was found to have normal mRNA and protein levels. This patient's clinical condition improved and he is alive at the age of 2.5 years. Thus we show that all patients with a classical clinical course of ARC had decreased expression of VPS33B whereas normal VPS33B expression was associated with good prognosis despite initial diagnosis of ARC. Children's Liver Disease Foundation (CLDF) ARC syndrome association Children Living with Inherited Metabolic Diseases (CLIMB) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) United States Department of Health & Human Services National Institutes of Health (NIH) - USA Birmingham Children's Hospital Research Foundation (BCHRF)

Enteropathogenic Escherichia coli (EPEC), an important paediatric diarrhoeal pathogen, employs multiple adhesins to colonize the small bowel and produces characteristic 'attaching and effacing' (A/E) lesions on small intestinal enterocytes. EPEC adhesins that have been associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP), EspA filaments and intimin. BFP are involved in bacteria-bacteria interaction and microcolony formation but their role in cell adhesion remains unclear; EspA filaments are components of the EPEC type III secretion system but since they interact directly with host cells they may also function as adhesins; intimin is the well characterized intimate EPEC adhesin which binds the translocated intimin receptor, Tir. However, other uncharacterized host cell receptors have been implicated in intimin-mediated adhesion. In this study, the role of BFP, EspA filaments and intimin in EPEC adhesion to intestinal brush border cells was assessed by observing adhesion of wild-type EPEC strain E2348/69 and a set of isogenic single, double and triple mutants in bfpA, espA and eae (intimin gene) to differentiated human intestinal Caco-2 cells. E2348/69 (bfpA(+) espA(+) eae(+)) adhered rapidly (10 min) to the brush border of Caco-2 cells and subsequently produced microcolonies and typical A/E lesions. Non-intimate brush border adhesion of double mutant strain UMD880 (bfpA(+) espA(-) eae(-)) also occurred rapidly, whereas adhesion of strain UMD886 (bfpA(-) espA(+) eae(-)) occurred later in the infection (1 h) and with much lower efficiency; confocal microscopy indicated BFP and EspA-mediated adhesion, respectively. Strain UMD883 (bfpA(-) espA(-) eae(+)), which is unable to translocate Tir, was non-adherent although this strain was able to form intimate attachment and A/E lesions when co-cultured with strain CVD206 (bfpA(+) espA(+) eae(-)) which supplied Tir to the membrane. Single mutant strains CVD206 (bfpA(+) espA(+) eae(-)) and UMD872 (bfpA(+) espA(-) eae(+)) showed adherence characteristics of strain UMD880 (bfpA(+) espA(-) eae(-)), whilst triple mutant strain UMD888 (bfpA(-) espA(-) eae(-)) was totally non-adherent. These results support an adhesive role for BFP and EspA in initial brush border cell attachment, and in typical EPEC which express both BFP and EspA filaments suggest a predominant role for BFP; EspA filaments, however, could serve as initial attachment factors in atypical EPEC which lacks BFP. The study found no evidence for an independent host cell intimin receptor or for other adhesive factors able to support bacterial adherence.

Nat. Genet. 42, 303–312 (2010); published online 28 February 2010; corrected after print 24 February 2011 In the version of this article initially published, the first and second paragraphs of the Results incorrectly stated that the C14ORF133 gene product was a previously unidentified protein and that no antibody against the C14ORF133 gene product (VIPAR) was available.