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1.
J Biol Chem ; 295(10): 3029-3039, 2020 03 06.
Article in English | MEDLINE | ID: mdl-31996372

ABSTRACT

Molybdenum cofactor (Moco) biosynthesis is a highly conserved multistep pathway. The first step, the conversion of GTP to cyclic pyranopterin monophosphate (cPMP), requires the bicistronic gene molybdenum cofactor synthesis 1 (MOCS1). Alternative splicing of MOCS1 within exons 1 and 9 produces four different N-terminal and three different C-terminal products (type I-III). Type I splicing results in bicistronic transcripts with two open reading frames, of which only the first, MOCS1A, is translated, whereas type II/III splicing produces MOCS1AB proteins. Here, we first report the cellular localization of alternatively spliced human MOCS1 proteins. Using fluorescence microscopy, fluorescence spectroscopy, and cell fractionation experiments, we found that depending on the alternative splicing of exon 1, type I splice variants (MOCS1A) either localize to the mitochondrial matrix (exon 1a) or remain cytosolic (exon 1b). MOCS1A proteins required exon 1a for mitochondrial translocation, but fluorescence microscopy of MOCS1AB variants (types II and III) revealed that they were targeted to mitochondria independently of exon 1 splicing. In the latter case, cell fractionation experiments displayed that mitochondrial matrix import was facilitated via an internal motif overriding the N-terminal targeting signal. Within mitochondria, MOCS1AB underwent proteolytic cleavage resulting in mitochondrial matrix localization of the MOCS1B domain. In conclusion, MOCS1 produces two functional proteins, MOCS1A and MOCS1B, which follow different translocation routes before mitochondrial matrix import for cPMP biosynthesis involving both proteins. MOCS1 protein maturation provides a novel alternative splicing mechanism that ensures the coordinated mitochondrial targeting of two functionally related proteins encoded by a single gene.


Subject(s)
Carbon-Carbon Lyases/metabolism , Mitochondria/metabolism , Alternative Splicing , Animals , COS Cells , Carbon-Carbon Lyases/genetics , Chlorocebus aethiops , Exons , Humans , Microscopy, Fluorescence , Mitochondrial Proteins/metabolism , Open Reading Frames/genetics , Organophosphorus Compounds/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Pterins/metabolism
2.
Biochem J ; 469(2): 211-21, 2015 Jul 15.
Article in English | MEDLINE | ID: mdl-26171830

ABSTRACT

Mammalian sulfite oxidase (SO) is a dimeric enzyme consisting of a molybdenum cofactor- (Moco) and haem-containing domain and catalyses the oxidation of toxic sulfite to sulfate. Following sulfite oxidation, electrons are passed from Moco via the haem cofactor to cytochrome c, the terminal electron acceptor. In contrast, plant SO (PSO) lacks the haem domain and electrons shuttle from Moco to molecular oxygen. Given the high similarity between plant and mammalian SO Moco domains, factors that determine the reactivity of PSO towards oxygen, remained unknown. In the present study, we generated mammalian haem-deficient and truncated SO variants and demonstrated their oxygen reactivity by hydrogen peroxide formation and oxygen-consumption studies. We found that intramolecular electron transfer between Moco and haem showed an inverse correlation to SO oxygen reactivity. Haem-deficient SO variants exhibited oxygen-dependent sulfite oxidation similar to PSO, which was confirmed further using haem-deficient human SO in a cell-based assay. This finding suggests the possibility to use oxygen-reactive SO variants in sulfite detoxification, as the loss of SO activity is causing severe neurodegeneration. Therefore we evaluated the potential use of PEG attachment (PEGylation) as a modification method for future enzyme substitution therapies using oxygen-reactive SO variants, which might use blood-dissolved oxygen as the electron acceptor. PEGylation has been shown to increase the half-life of other therapeutic proteins. PEGylation resulted in the modification of up to eight surface-exposed lysine residues of SO, an increased conformational stability and similar kinetic properties compared with wild-type SO.


Subject(s)
Amino Acid Metabolism, Inborn Errors/drug therapy , Enzyme Replacement Therapy , Oxidoreductases Acting on Sulfur Group Donors , Oxygen , Sulfite Oxidase/deficiency , Amino Acid Metabolism, Inborn Errors/genetics , Amino Acid Metabolism, Inborn Errors/metabolism , Animals , Enzyme Stability/genetics , HEK293 Cells , Heme/chemistry , Heme/genetics , Heme/metabolism , Humans , Hydrogen Peroxide , Mice , Oxidoreductases Acting on Sulfur Group Donors/chemistry , Oxidoreductases Acting on Sulfur Group Donors/genetics , Oxidoreductases Acting on Sulfur Group Donors/metabolism , Oxidoreductases Acting on Sulfur Group Donors/therapeutic use , Oxygen/chemistry , Oxygen/metabolism , Oxygen Consumption/drug effects , Polyethylene Glycols/chemistry , Sulfite Oxidase/genetics , Sulfite Oxidase/metabolism
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