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1.
Nat Commun ; 7: 11601, 2016 05 27.
Article in English | MEDLINE | ID: mdl-27231142

ABSTRACT

Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism-dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.


Subject(s)
Cation Transport Proteins/genetics , Dystonic Disorders/genetics , Homeostasis , Manganese/metabolism , Mutation , Parkinsonian Disorders/genetics , Adolescent , Animals , Cation Transport Proteins/metabolism , Child , Child, Preschool , Dystonic Disorders/metabolism , Female , Genetic Predisposition to Disease/genetics , HEK293 Cells , Humans , Male , Manganese/blood , Parkinsonian Disorders/metabolism , Pedigree , Young Adult , Zebrafish/embryology , Zebrafish/metabolism
2.
Biochim Biophys Acta ; 1862(1): 135-44, 2016 01.
Article in English | MEDLINE | ID: mdl-26529285

ABSTRACT

INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.


Subject(s)
Leptin/metabolism , Liver Cirrhosis/metabolism , Liver/pathology , Non-alcoholic Fatty Liver Disease/metabolism , Osteopontin/metabolism , Animals , Cell Line , Cells, Cultured , Gene Deletion , Hepatocytes/metabolism , Hepatocytes/pathology , Leptin/genetics , Liver/metabolism , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Male , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/pathology , Osteopontin/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats, Sprague-Dawley , Signal Transduction , Up-Regulation
3.
J Bacteriol ; 192(22): 6093-8, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20833800

ABSTRACT

Here we show that the type III secretion gatekeeper protein SepL resembles an aberrant effector protein in binding to a class 1 type III secretion chaperone (Orf12, here renamed CesL). We also show that short N-terminal fragments (≤70 amino acids) from SepL are capable of targeting fusion proteins for secretion and translocation.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Molecular Chaperones/metabolism , Amino Acid Sequence , Escherichia coli Proteins/genetics , Molecular Sequence Data , Protein Binding , Protein Sorting Signals , Protein Transport , Sequence Alignment , Sequence Homology, Amino Acid
4.
Proc Natl Acad Sci U S A ; 103(40): 14941-6, 2006 Oct 03.
Article in English | MEDLINE | ID: mdl-16990433

ABSTRACT

Several pathogenic strains of Escherichia coli exploit type III secretion to inject "effector proteins" into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of >60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into >20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in >20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage "metagenome," acting as a crucible for the evolution of pathogenicity.


Subject(s)
Bacteriophage lambda/metabolism , Escherichia coli O157/metabolism , Escherichia coli Proteins/metabolism , Chromosomes, Bacterial/genetics , Genome, Bacterial/genetics , Humans , Prophages/genetics , Sequence Homology , Shigella/metabolism
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