Analysis of the cellular roles of MOCS3 identifies a MOCS3

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Analysis of the cellular roles of MOCS3 identifies a MOCS3independent localization of NFS1 at the tips of the centrosome Yannika Neukranz, Annika Kotter, Lena Beilschmidt, Zvonimir Marelja, Mark Helm, Ralph Gräf, and Silke Leimkühler Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01160 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Biochemistry

Analysis of the cellular roles of MOCS3 identifies a MOCS3-independent localization of NFS1 at the tips of the centrosome Yannika Neukranz1,2, Annika Kotter3, Lena Beilschmidt1, Zvonimir Marelja1,4, Mark Helm3, Ralph Gräf2* and Silke Leimkühler1* From the 1Department of Molecular Enzymology and 2Cell Biology, Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany; 3Institute of Pharmacy and Biochemistry, Johannes Gutenberg-Universität Mainz, 55128 Mainz; Germany. 4current

address: Imagine Institute, Université Paris Descartes-Sorbonne Paris Cité, Paris, France

*Corresponding

Authors

Silke Leimkühler; Department of Molecular Enzymology, Institute of Biochemistry and Biology, and Ralph Gräf, Department of Cell Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany; Tel.: +49-331-977-5603; Fax: +49-331-977-5128; E-mails: [email protected]; [email protected]

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ABSTRACT

The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the L-cysteine desulfurase NFS1, MOCS3 and MOCS2A. Human MOCS3 is a dualfunction protein that was shown to play an important role in Moco biosynthesis and in the mcm5s2U thio-modifications of nucleosides in cytosolic tRNAs for Lys, Gln and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, based on the absence of Moco in these cells. Further, mcm5s2U thio-modified tRNAs were not detectable. Since the L-cysteine desulfurase NFS1 was shown to act as sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods we identified a MOCS3-independent novel localization of NFS1 at the centrosome.


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Biochemistry

INTRODUCTION Molybdenum is an essential trace element for humans. In enzymes, molybdenum is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT), and after molybdenum ligation the molybdenum cofactor (Moco) is formed.1 Moco is present in five enzymes in humans: sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase 1 and two mitochondrial amidoxime reducing components, mARC1 and mARC2.2, 3 Sulfite oxidase is the only one of the five molybdoenzymes which is essential for humans.4, 5 Human Moco deficiency leads to the pleiotropic loss of all these molybdoenzymes and usually progresses to death at an early age.6, 7 The deficiency of Moco is an autosomal recessive disease and its deficiency in patients has been described for the first time by Duran et al. in 1978.4 Patients with Moco deficiency are in general seriously affected in the neonatal period and exhibit symptoms that include severe neurological abnormalities like seizures, dislocated ocular lenses, skeletal changes, dysmorphic features, and intellectual disability.8 Genes and the encoded proteins were named in humans as MOCS (molybdenum cofactor synthesis).9 In humans, six proteins that directly catalyze Moco biosynthesis have been identified: MOCS1A, MOCS1B, MOCS2A, MOCS2B, MOCS3 and Gephyrin (GPHN).9 Up to now, the majority of patients have been described with mutations in MOCS1, MOCS2, or GPHN.10 Mutations in MOCS1 are the predominant ones effecting two-thirds of the described cases.11 Recently, one patient with a homozygous mutation in MOCS3 was identified that showed only mild abnormalities in sulfite metabolism.12 Genomic analysis revealed a missense mutation leading to the amino acid substitution A257T in the highly conserved Nterminal domain of the protein. Moco biosynthesis starts with the conversion of 5'GTP to cyclic pyranopterin monophosphate, a reaction catalyzed by the proteins MOCS1A and MOCS1B in the mitochondrial matrix of human


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cells.13-15 cPMP is further transferred to the cytosol where it is converted to MPT by insertion of two sulfur atoms, forming the dithiolene group which ligates the molybdenum atom.16, 17 The step of sulfur insertion is catalyzed by MPT synthase, a protein consisting of MOCS2A and MOCS2B. MOCS2A thereby binds the sulfur atom in form of a C-terminal thiocarboxylate group.18 MOCS2B is involved in binding of cPMP during the reaction.19 The molybdenum atom is inserted into the MPT backbone by GPHN.20, 21 The regeneration of the C-terminal thiocarboxylate group on MOCS2A after MPT formation involves the MOCS3 protein.22, 23 MOCS3 structurally and functionally resembles the ubiquitin activating enzyme E1 and activates MOCS2A, a protein with a ubiquitin-fold, through its N-terminal MoeB-like domain by adenylation.24 The C-terminus of MOCS3 has a rhodanese-like fold and was shown to carry the sulfur to be transferred to MOCS2A in form of a persulfide group.23 The sulfur is mobilized from the cytosolic form of the L-cysteine desulfurase NFS1, a pyridoxal-phosphate dependent L-cysteine desulfurase, which forms a persulfide group on its conserved Cys381 residue.25, 26 The persulfide group is further transferred to Cys412 on the C-terminal rhodanese-like domain of MOCS3. MOCS3 has been further revealed to be a bi-functional protein in eukaryotes.27 It has been shown that human MOCS3 not only activates MOCS2A, but also the ubiquitin related modifier 1 (URM1).28 URM1 was identified to be involved in sulfur transfer for the formation of 2-thiouridine in tRNA.29, 30 So far in humans, tRNAs were identified to contain 5-methoxy-carbonylmethyl-2-thiouridine (mcm5s2U34) and 2methylthio-N6-threonylcarbonyl-adenosine (ms2t6A37) thiomodifications in cytoplasmic tRNAs or 2-methylthio-N6-(cis-hydroxyisopentenyl)-adenosine (ms2io6A37) and 5-taurinomethyl-2thiouridine (τm5s2U34) thiomodifications in mitochondrial tRNAs.31-35 In humans, it was shown that the proteins MOCS3, URM1, CTU1 and CTU2 are essential for cytosolic s2U34 formation, while proteins of the elongator complex (ELP) synthesize the mcm5-group.29 For the formation of


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Biochemistry

the thiocarboxylate (-COSH) group at the C-terminal glycine of URM1, the MOCS3-bound persulfide is directly transferred to URM1.36 As described above, also for this pathway MOCS3 receives its sulfur from the cytosolic form of NFS1.25 NFS1 has been intensively studied for its role in Fe-S cluster biosynthesis.37 In humans, the main proteins required for Fe-S cluster biosynthesis are NFS1, ISD11, ISCU and Frataxin (FXN), which form the quaternary core complex.38-40 ISD11 thereby is exclusively present in eukaryotes and was described as a stabilizing factor for NFS1, being essential for the activity in Fe-S cluster formation in mitochondria.41 After the synthesis of the Fe-S cluster on ISCU by the additional help of chaperones, the cluster is transferred by the help of carrier proteins to mitochondrial acceptor proteins.42 The export of a “sulfur compound” from mitochondria has been proposed to be required for the cytosolic Fe-S cluster assembly (CIA) pathway.43 After Fe-S cluster formation in the cytosol by the complex CIA machinery, Fe-S clusters are transferred to cytosolic and nuclear acceptor proteins, with the help of NUBP1, NUBP2, NARFL and CIAO1 proteins of the CIA pathway.44 However, the mammalian Fe-S core complex components have also been identified in the cytosol42, 45-48, suggesting that Fe-S cluster biogenesis assembly may independently operate in parallel in mitochondria and the cytosol. It has been shown that cytosolic NFS1 is an active Lcysteine desulfurase, which forms a complex with the cytosolic isoform of ISCU.49 Further, the cytosolic form of NFS1 has been shown recently to contribute to de novo Fe-S synthesis in the cytosol and to the functionalization of mammalian cytosolic Fe-S proteins.50 However, cytosolic NFS1 has the additional role apart from Fe-S cluster assembly described above with its involvement in sulfur transfer for Moco biosynthesis and tRNA thiolation, two pathways which require only low amounts of the protein.


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In this report we constructed the first homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. We analyzed the effect of the deletion of MOCS3 on Moco biosynthesis and the thio-modification of tRNAs. We show that in addition to an impaired tRNA thiolation, sulfite oxidase activity was largely affected in the constructed MOCS3 knockout cell lines, leading to the degradation of apo-sulfite oxidase in the cytosol. Since MOCS3 and NFS1 interact in the cytosol, we analyzed the effect of the absence of MOCS3 on the cytosolic localization of NFS1. By different methods, we detected a novel localization of NFS1 at the centrosome.

MATERIALS AND METHODS Cultivation of Mammalian Cell lines HEK293T and HeLa (DSMZ) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, PAN-Biotech, Germany) supplemented with 10% fetal bovine serum (FBS, PANBiotech, Germany) and 2 mM L-Glutamine. The cells were maintained at 37°C and 5% CO2 adherently in T75 or T25 cell culture flasks (Sarstedt) until 80% confluent. The cells were detached via Trypsin/EDTA (Gibco, Life Technologies) and passaged every 3 to 4 days.

Generating MOCS3 knockout cells with CRISPR/Cas9 The CRISPR/Cas9 method was used to generate stable MOCS3 knockout cell lines. The protocol was adapted after Ran et al.51 The method is based on a complementary gRNA to target the gene of interest providing a cleavage site for the Cas9 nuclease. The cleaved DNA is repaired by error prone non-homologous end joining (NHEJ) leading to deletions, insertions or frame-shifts preferentially resulting in loss of function mutations. gRNAs complementary to DNA near the start


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Biochemistry

codon of the MOCS3 gene were designed using the MIT software (http:// tools.genomeengineering.org).51 The forward guide and the reverse guide were annealed using a standard protocol. They were constructed with BbsI restriction sites to enable cloning into the pSpCas9(BB)2A-Puro vector (Addgene). This vector already contains the scaffolding part of the gRNA and the gene for Cas9. The resulting plasmid was transiently transfected into the HEK293T cells. Positively transfected cells were selected with puromycin. Single cells were grown into colonies and analyzed via sequencing (GATC) and immunoblotting using an -MOCS3 antibody (Abcam).

Immunoblotting Whole cell lysates (25 - 50 μg) or the centrosomal fractions were separated by SDS-PAGE and transferred onto a PVDF membrane (AmershamTM HybondTM, GE Healthcare). Protein transfer was performed using Mini-Protean 2 Cell chambers (BioRad). The primary antibodies α-MOCS3 (1:4000, Abcam), α-NFS1 (1:1000, Abcam), α-γ-tubulin (1:5000, Sigma), α-Cdk5Rap2 (1:500), α-aconitase 2, mitochondrial (1:2500, Abcam ab110321), α-ICDH (1:5000, Abcam ab172964), αsulfite oxidase (1:1000, Abcam), α-laminB1 (1:2000, Abcam) and α-γ-actin (1:7500, Sigma) were used for protein detection together with the peroxidase-coupled secondary antibodies (α-rabbit POD, 1:10000, Sigma) (α-mouse POD, 1:5000, Sigma). The Blots were developed with chemiluminescence via the Fusion SL Vilber Lourmat (peqlab) imaging system.

MTT Assay Dissolved MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is converted to insoluble purple formazan by dehydrogenases in living cells. Formazan can be solubilized by


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isopropanol and measured spectrophotometrically. 10x103 cells/well were seeded in a 96 well plate. 50µl of MTT solution was added to each well and incubated for 3 hrs at 37°C. Subsequently, 150µl of MTT solvent was added and the MTT formazan was detected after 15 min at 590nm.

Isocitrate Dehydrogenase Activity Assay The activity of isocitrate dehydrogenase (ICDH) was measured as general house-keeping control protein.42 HEK293T cells were grown in T75 culture flasks until 90% confluent. The cells were harvested and lysed in non-denaturing lysis buffer (50 mM Tris-HCL, 1% NP-40, pH 8). The protein concentration was determined using Bradford reagent. To measure the ICDH activity, 25 µl of cell lysate was added to 225 µl reaction buffer (50mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, 0.5 mM NADP+, pH 8). After an incubation of 1 min at 37°C 250µl of starting reaction (50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, 0.5 mM NADP+, 5 mM isocitrate, pH 8) was added and the reduction of NADP+ was followed for 3 min at 340 nm. The specific activity was determined using an extinction coefficient of NADPH (ε340nm = 6220 mM-1). Aconitase Activity Assay Aconitase is a [4Fe-4S] cluster containing protein that catalyzes the isomerization of citrate to isocitrate via cis-aconitate. HEK293T cells were grown in T75 culture flasks until 90% confluency. The cells were harvested and lysed in non-denaturing lysis buffer (50 mM Tris-HCL, 1% NP-40, pH 8). The protein concentration was determined via a Bradford assay. The aconitase activity was measured using 50 µl of cell lysate mixed with 250 µl reaction buffer (50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, 0.5 mM NADP+, 0.05 U isocitrate-dehydrogenase (Sigma), pH 8). This was incubated for 5 min at 37°C before the addition of 200 µl staring buffer (50mM Tris-HCl, 50 mM


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Biochemistry

NaCl, 5 mM MgCl2, 2.5 mM cis-aconitate. The reaction is followed at 340 nm by the reduction of NADPH as ICDH converts the product of aconitase. The specific activity was calculated using the extinction coefficient of NADPH (ε340nm = 6220 mM-1).

Sulfite Oxidase Activity Assay The activity assay of the Moco dependent enzyme sulfite oxidase was adapted from Johnson et al. (1991).52 Cells were grown in a T75 cell culture flask until 90% confluent. They were harvested and the pellet resuspended with extraction buffer (50 mM Tris-acetate, 0.1 mM EDTA and 1% NP40, pH 8.5). The probes were vortexed and centrifuged (12000 g, 15 min, 4°C) to obtain the cell lysate. The protein concentration was determined with Bradford reagent. The enzyme activity was measured using 150 μl of cell lysate in a total reaction volume of 1 ml. The reaction buffer consisted of 800 μl of 50 mM Tris-acetate, 0.1mM EDTA and 1% NP-40, pH 8.5 to which 10 μl of 17 mM sodium deoxycholic acid, 5 μl of 10 μM potassium cyanide, 33 μl of cytochrome c (6 mg/ml) and 2 μl of 100 mM sodium sulfite were added. The reduction of cytochrome c was monitored at 550 nm for 5 min. The specific activity using the extinction coefficient of cytochrome c (ε550nm = 19.36 M-1) was calculated.

tRNA Extraction 48 hours before harvesting the cells were seeded on 3 x T75 cell culture flasks until 90% confluent. The cells were taken up with TriFast (Peqlab) and a 1:5 volume of chloroform was added. After centrifugation the upper, aqueous phase was transferred into a new falcon and precipitated with 1 times the volume of isopropanol overnight at -20°C. The samples were spun down (13000 g, 1.5 h,


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4°C) and the resulting pellet was washed 3 x with 70% ethanol. The pellet was dried at 37°C for 10-15 min. The precipitated RNA was dissolved in 100 μl 0.3M NaOAc, pH 4.5 for 15 min at 55°C. 100 μg of total RNA per gel are separated by 10% urea-polyacrylamide gel electrophoresis (PAGE) run at 200V for 1 hr and 15 min. Subsequently the gels were stained with ethidium bromide solution and the tRNA bands were cut out and placed in crush-n-soak buffer (50mM sodium acetate, 150mM sodium chloride, pH 7.0) at 4°C overnight to release the RNA out of the gel. This was then precipitated overnight at -20°C with a 1:1 dilution with isopropanol before being washed twice with 70% ethanol. The tRNA pellets were dried at 37°C for 10-15 min. They were dissolved in 50μl 0.3 M NaOAc for approximately 15 min at 55°C. HPLC analysis was performed as described by Gehrke and Kuo.53

LC-MS 5 µg total tRNA was digested into nucleosides using 0.6 U nuclease P1 from P. citrinum (SigmaAldrich), 0.2 U snake venom phosphodiesterase from C. adamanteus (Worthington), 2 U FastAP (Thermo Scientific), 10 U benzonase (Sigma-Aldrich), 200 ng pentostatin (Sigma-Aldrich) and 400 ng tetrahydrouridine (Merck-Millipore) in 25 mM ammonium acetate (pH 7.5; SigmaAldrich) over night at 37°C. Technical duplicates with 1 µg digested tRNA were analyzed via LCMS (Agilent 1260 series and Agilent 6460 Triple Quadrupole mass spectrometer equipped with an electrospray ion source (ESI)). The solvents consisted of 5 mM ammonium acetate buffer (pH 5.3; solvent A) and LC-MS grade acetonitrile (solvent B; Honeywell). The elution started with 100% solvent A with a flow rate of 0.35 ml/min, followed by a linear gradient to 8% solvent B at 10 min, 40% solvent B at 20 min. Initial conditions were regenerated with 100% solvent A at 23 min for another 7 min. The column used was a C18 Synergi Fusion (4 µM particle size, 80 Å pore


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Biochemistry

size, 250 × 2.0 mm; Phenomenex). The UV signal at 254 nm was recorded via a diode array detector (DAD) to monitor the four canonical nucleosides. ESI parameters were as follows: gas temperature 350°C, gas flow 8 l/min, nebulizer pressure 50 psi, sheath gas temperature 300°C, sheath gas flow 12 l/min, capillary voltage 3500 V. The MS was operated in the positive ion mode using Agilent MassHunter software in the dynamic MRM (multiple reaction monitoring) mode. For quantification, the amounts of mcm5U and mcm5s2U were calculated by means of a calibration curve and then normalized to the UV signal of uridine for inter-sample comparison.

Quantification of Moco and cPMP in HEK293T Cells Moco and cPMP can be oxidized into their fluorescent degradation products FormA and Compound Z, respectively. These were then eluted via QAE chromatography and quantified via HPLC.54 The HEK293T cells were grown in two T75 cell culture flasks until 90% confluent. They were harvested and resuspended in 800 µl chilled 100mM Tris-HCl (pH 7.2) following cell lysis through sonification (on 2 sec, off 2 sec, 20%, 45 sec). The protein concentrations were determined via Bradford reagent. For both, Moco and cPMP, 50 µl of solution A (1063 µl I2/KI and 100µl 37% HCl) was added to 400µl cell lysate followed by the addition of 150 µl of I2/KI solution turning the samples dark brown. They were kept in the dark overnight. Subsequently, 100µl of 1% ascorbic acid was added to 400 µl supernatant after centrifugation. This was followed by the addition of 200 µl of 1M Tris to change the pH to 8.3. The cPMP sample was then ready for column loading. The FormA samples need to be dephosphorylated and are thereby treated with 30 µl of 1M MgCl2 and 2 µl of fast alkaline phosphatase which is incubated for two hours. Purification of FormA and CompoundZ was performed using QAE chromatography. FormA elution requires 10 mM acetic acid from which 9 fractions are collected (500 µl). The cPMP samples were eluted


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with 100 mM HCl collecting 9 times 500 µl fractions. Subsequently, the fractions were loaded onto the HPLC and quantified after separation on a reversed phase C18 column.

Isolation of Centrosomes The centrosomes were isolated via sucrose density gradients. The protocol was adapted from Reber et al. (2011).55 HEK293T cells were grown on 15 24.5 x 24.5 cm plates and until nearly confluent. Nocodazole (10 μg/ml) and cytochalasin A (5 μg/ml) were added to the medium for 90 min. Next, the medium is aspirated and the cells washed with PBS, 8% sucrose in 0.1xPBS, 8% sucrose in ddH20 and finally 1 mM Tris, pH 8.0, 8 mM 2-mercaptoethanol. Subsequently the cells are incubated in lysis buffer for 20min. The cell lysates were combined and centrifuged at 1500xg for 3 min at 4°C. The supernatant was layered on 80% sucrose cushions onto which the centrosomes will sediment during centrifugation (25000 g, 15 min at 4°C). 2 ml of the solution above the cushions were collected and the centrosomes further purified by discontinuous sucrose gradient, 5 ml 70%, 3 ml 50% and 3 ml 40% sucrose solutions (130000 g, 90 min at 4°C). 500 μl fractions were collected ranging from sucrose concentrations of 40-60%.

Immunofluorescence Microscopy The centrosomal fractions were diluted (1:50) in 10mM Pipes buffer (pH7.2) and centrifuged (3000rpm, 30 min, 4°C) onto the poly-L-Lysin-coated cover slips. For in vivo localization studies, HEK293T and HeLa cells were grown on coverslips. Both the cells and the isolated centrosomes were fixed with methanol (5 min at -20°C) and subsequently washed three times with PBS. Furthermore, the cells were incubated with blocking solution (0.5% BSA in PBS) for 40 minutes.


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Biochemistry

The dilutions for the 1st antibodies were prepared in PBS (α-NFS1, 1:200, Abcam) (α-γ-tubulin, 1:800, sigma) (GT335, 1:500). The cells were incubated overnight with the 1st antibody solution followed by three more washing steps with PBS. Next, the second antibody solution was added onto the coverslips for one hour (Alexa Flour 488 goat α-rabbit IgG, 1:200, Invitrogen) (Alexa Flour 568 goat α-mouse IgG, 1:200, Invitrogen). The cells were washed three times with PBS. The coverslips were placed on the objective slide with Mowiol with DAPI (1:1000, Sigma). Images were conducted using the Zeiss LSM710 (Carl Zeiss Microscopy GmbH, Jena, Germany) laser scanning confocal microscope equipped with an PlanApo 1.4/63x oil objective or via wide-field microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany) using a Zeiss CellObserver HS System with a PlanApo 1.4/100x objective. In the latter case image stacks were deconvolved with Axiovision 4.8 using the iterative method and a measured PSF.56


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RESULTS Generating HEK293T MOCS3 knockout cell lines with the CRISPR/Cas9 system. HEK293T cells (DSMZ) were used to generate the MOCS3 knockout cell line. HEK293T cells have a near triploid karyotype but contain only two copies of chromosome 20 on which the MOCS3 gene is localized. gRNAs were designed complementary to the beginning of the MOCS3 gene, which preferentially lead to random mutations with a desired premature termination of MOCS3 transcription. DNA sequencing revealed the mutations in the alleles and identified one heterozygous (+/-) cell line and one homozygous (-/-) MOCS3 knockout cell lines (Figure 1B and Supplementary Figure S1 and Supplementary Figure S2). Additionally, the absence of the MOCS3 protein in the knockout cell line was confirmed by immunodetection using a MOCS3 specific antibody. The results in Figure 1A show that in the homozygous (-/-) cell lines, MOCS3 was not detected while in the heterozygous (+/-) cell line the presence of the MOCS3 protein was largely reduced in comparison to the MOCS3 wild-type HEK293T cells. Thus, while one allele still had a functional MOCS3 coding sequence in the (+/-) cell lines, the expression nevertheless was not sufficient to produce comparable MOCS3 levels as present in the wild-type cells. Therefore, both MOCS3 alleles seem to be required to supply the necessary MOCS3 levels. The MTT assay was applied to analyze for changes in the cell growth caused by the absence of MOCS3. The results in Figure 1C show that the absence of MOCS3 has an impact on cell division. The cells were able to divide, but the growth rate revealed to be significantly reduced down to 50% in the homozygous (-/-) as well as in the heterozygous (+/-) cell lines compared to the wild type cells. Thus, the reduced amount of MOCS3 in the (+/-) cells is not sufficient to maintain normal cell division, revealing the importance of MOCS3 in this process.


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Biochemistry

Figure 1: Generation of MOCS3 knockout cell lines using the CRISPR/Cas9 system. (A) Immunodetection of MOCS3 in different HEK293T cell lines. The generated HEK293T CRISPR/Cas9 cell lines with a homozygous (-/-) and heterozygous (+/-) knockout in MOCS3 were analysed for the presence of the MOCS3 protein by immunodetection. The cell lines were lysed with NP-40 buffer and separated by 12% SDS-PAGE. The proteins were transferred on a PVDF membrane and detected with antibodies against MOCS3. Beta-actin was used as a loading control. (B) Schematic diagram representing the two alleles of the MOCS3 gene and the loss of function mutations in each cell line. The genomic DNA was isolated and PCR fragments amplified including the cutting site of the Cas9 protein. These were then sequenced and the two alleles identified. The sequences are presented in Supplementary Figure S1. (C) Cell proliferation was measured using the MTT assay. 10x103 cells/well were seeded in 96 well plates. 50µl of MTT solution was added to each well and incubated for 3 hrs at 37°C. Subsequently, 150 µl of MTT solution was added and after 15 min incubation the absorbance was determined at OD590. The effect of the MOCS3 knockout on sulfite oxidase activity. MOCS3 is essential for the biosynthesis of Moco catalyzing the formation of the thiocarboxylate group on MOCS2A.22 Consequently, the activity of molybdoenzymes are expected to be impaired in MOCS3 deficient cell lines. We selectively measured sulfite oxidase activity as representative


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molybdoenzyme. The results in Figure 2A show that in the homozygous (-/-) MOCS3 knockout cell line only a background sulfite oxidase activity of about 10% of the wild type cells was detected. This background activity which is not based on sulfite oxidase activity was somehow expected, since cell extracts were measured that also contain other cytochrome c-reducing enzymes. In comparison, the sulfite oxidase activity in the heterozygous (+/-) MOCS3 cell line was not affected, still containing one functional allele for MOCS3. Here, even the low levels of MOCS3 present in the (+/-) cells are sufficient to maintain the levels of sulfite oxidase activity. Therefore, all Moco produced in the MOCS3 (+/-) cells (see below) likely is inserted into sulfite oxidase to ensure enough active enzyme. This might be based on the cellular importance of sulfite oxidase in sulfite detoxification. The sulfite oxidase activity of the homozygous (-/-) MOCS3 knockout could be rescued by reintroducing the MOCS3 protein (as eCFP-tagged fusion protein57), which resulted in 80% increase in activity. Here, an incomplete compensation of sulfite oxidase activity might be caused by a low transfection efficiency of 40% (Figure 2B). Separate introduction of the N-terminal MOCS3 MoeB-like domain or only the C-terminal MOCS3 rhodanese-like domain as eCFP-tagged fusion proteins, respectively, did not result in a detectable increase in sulfite oxidase activity (Figure 2A). Conclusively, both domains of MOCS3 are essential to fulfil the role in the formation of the thiocarboxylate group on MOCS2A, as reported before. Here, the N-terminal domain is essential to activate the C-terminus of MOCS2A in an ATP-dependent manner and the C-terminal rhodanese-like domain provides the sulfur for the formation of the thiocarboxylate group. The activities of other molybdoenzymes like xanthine dehydrogenase or aldehyde oxidase could not be tested, since both enzymes are only present in low amounts in HEK293T cells and their activities were below the limit of detection (data not shown).


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The protein levels of sulfite oxidase and actin were also determined by immunodetection using specific antibodies raised against these proteins (Figure 2C). Immunodetection of sulfite oxidase showed a band that was detected in the wildtype cells and the heterozygous cell line (+/-), but not in the homozygous (-/-) MOCS3 knockout cells. A slightly reduced band for sulfite oxidase was detected in the transfected cells with the eGFP-MOCS3 expression construct. The reduced protein amount correlated with the 80% activity of sulfite oxidase detected in these cells. No band corresponding to sulfite oxidase protein was obtained in the transfected cells with the eCFP-tagged MoeB-like domain or the eCFP-tagged rhodanese-like domain of MOCS3 (Figure 2C).

Figure 2: Sulfite oxidase activity in MOCS3 knockout cell lines. (A) Sulfite oxidase activity was determined by the reduction of cytochrome c at 550 nm (n=4). HEK293T wild type, heterozygous (+/-), homozygous (-/-) MOCS3 knockout cell lines or


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homozygous (-/-) MOCS3 cell lines transfected with plasmids encoding eCFP-MOCS3, eCPFMoeBD or eCFP-RLD were lysed with NP-40 buffer. The reaction contained sodium sulfite (substrate), cyanide and Na-deoxycholate. The P-values were calculated using an unpaired t-test (ns P>0.05, *** P≤0.001, **** P≤0.0001). (B) Complementation of homozygous (-/-) MOCS3 knockout cell lines with eCFP-MOCS3. MOCS3 knockout cells (-/-) were transfected with an eCFP-MOCS3 plasmid and subsequently fixed onto cover slips and eCFP fluorescence was detected using a wide field microscope. DAPI (blue) was used to stain the nucleus. (C) Immunodetection of sulfite oxidase in different HEK293T cell lines. The generated HEK293T CRISPR/Cas9 cell lines with a homozygous (-/-) and heterozygous (+/-) knockout in MOCS3 were analysed for the presence of the sulfite oxidase protein by immunodetection. The cell lines were lysed with NP-40 buffer, loaded onto a 12% gel and separated by SDS-PAGE. The proteins were blotted on a PVDF membrane and incubated with antibodies against sulfite oxidase. Actin was used as a loading control.

The effect of the MOCS3 knockout on Moco biosynthesis. Since MOCS3 effects the conversion of cytosolic cPMP to MPT in the second step of Moco biosynthesis, we expected an accumulation of cPMP in the MOCS3 knockout cells. cPMP can be detected after oxidation to its fluorescent product Compound Z, which can be quantified after separation on a reversed phase C18 column. As expected, the results in Figure 3A show a two to three-fold increase in cPMP accumulation in the MOCS3 knockout cells in comparison to the wild type and the heterozygous cell line, which showed the same level of cPMP accumulation. However, already the wild-type cells accumulated some cPMP, which might indicate that the export of cPMP from the mitochondria to the cytosol is the rate limiting step in the reaction of MPT production, which is catalyzed in the cytosol. We also quantified MPT levels in the MOCS3 knockout cells in comparison to the wild type. MPT and Moco can be quantified as FormA after oxidation to their fluorescence derivative FormA by potassium iodide. For the homozygous MOCS3 mutant, no FormA was detected, while the heterozygous mutant strain showed an about


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Biochemistry

three-fold lower level of FormA in comparison to the wild-type strain (Figure 3B). This result is consistent with the lower levels of MOCS3 in the (+/-) cell lines.

Figure 3: Quantification of cPMP and Moco in MOCS3 knockout cell lines. (A) The cPMP content of HEK293T wild type and heterozygous (+/-) or homozygous (-/-) MOCS3 knockout cell lines was determined after conversion of cPMP to Compound Z by overnight oxidation with acidic iodine. (B) The Moco content of the indicated cell lines was quantified after conversion of Moco to FormA by overnight oxidation with acidic iodine. cPMP and FormA were separated by QAE ion exchange chromatography and applied on a reversed phase C18 column. Elution of Compound Z or FormA was monitored with an Agilent 1100 series fluorescence detector with excitation at 383 nm and emission at 450 nm. The total Compound Z or FormA content was normalized to the total protein concentration determined by Bradford. The P-values were calculated using an unpaired t-test (n = 3; ns P>0.05, * P≤0.05). The Effect of the MOCS3 knockout on tRNA thiolation. It has been reported previously, that MOCS3 is involved in mcm5s2U34 tRNA modifications in the cytosol by forming a thiocarboxylate group on URM1, which is further used as sulfur providing protein for the tRNA nucleoside modification.28 To confirm the involvement of MOCS3 in this reaction, the mcm5s2U modified nucleosides were quantified. tRNAs were isolated via phenol/chloroform phase separation and subsequently separated by urea-PAGE from which the


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corresponding bands were excised. Following solubilization, the modified nucleosides were separated and quantified after the method described by Gehrke and Kuo, (1989).53 The obtained peak areas were normalised to the pseudouridine peak. The results show that in the knockout cell line (-/-) the modified mcm5s2U was not detected, with the simultaneous accumulation of only mcm5U modified nucleosides (Figure 4A). The identity and relative levels of the mcm5s2U and mcm5U nucleosides was confirmed by mass spectrometry (Figure 4B). In the heterozygous MOCS3 knockout cell line a small amount of the mcm5U precursor was detected, while it was undetectable in wild type HEK 293T. This low accumulation of the mcm5U precursor might be based on the low levels of MOCS3 in the (+/-) cells which, however, are sufficient to maintain almost the levels of the modified mcm5s2U as wild-type levels. This might indicate that the overall cytosolic levels of the mcm5s2U modification is very low.


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Figure 4: Quantification of mcm5s2U tRNA thiomodifications in MOCS3 knockout strains. (A) Total RNAs from HEK293T wild type and heterozygous (+/-) or homozygous (-/-) MOCS3 knockout cell lines were extracted with the Trifast reagent and separated by 10% Urea-PAGE. tRNAs were digested into nucleosides and quantified after separation on a C18 reversed phase column by HPLC chromatography. The peak corresponding the mcm5s2U modified nucleosides or the mcm5U precursor were quantified by their absorbance at 274 nm. The corresponding peaks were integrated and normalized to the internal pseudouridine standard. The P-values were calculated using an unpaired t-test (n = 2; ns P>0.05, ** P≤0.01). (B) tRNA samples were analysed by LC-MS. The effect of the absence of MOCS3 on isocitrate and aconitase activities. The activity of isocitrate dehydrogenase (ICDH) was measured to elucidate the effect of the loss of MOCS3 on the general metabolism of the cells. ICDH is an enzyme of the citric acid cycle that catalyzes the conversion of isocitrate to α-ketoglutarate and CO2. Three ICDH enzymes are present in the cell. However, we analyzed the activities of ICDH in whole extracts of HEK293T cells, so


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that we measured the overall activities of all three enzymes. The enzymes do not contain any cofactors or metals for catalysis. The results in Figure 5A show no significant changes in ICDH activities caused by the absence of MOCS3, with only slightly reduced enzyme activity of about 10-20% in both the homozygous and heterozygous cells in comparison to the wild type cells. The protein levels for ICDHs in the heterozygous and homozygous cell lines were also similar to that of the wild type HEK293T cells (Figure 5C). Additionally, the [4Fe-4S] cluster containing enzyme aconitase was analyzed for effects on its activity caused by the absence of MOCS3. Two aconitases are present in the cell, one in mitochondria and one in the cytosol. Here, we measured the overall cellular aconitase activities and did not separate the extract into a cytosolic and mitochondrial fraction. The results in Figure 5B show an approximately 35% reduction in aconitase activity obtained in the homozygous (-/-) MOCS3 knockout cells. The same levels of reduction were also observed in the heterozygous MOCS3 (+/-) cells. The protein expression levels (here only the mitochondrial isoform was detected with the antibody used) were almost comparable in all three cell lines (Figure 5C). This implies that the reduced aconitase activities in both the MOCS3 (-/-) and (+/-) cell lines have an effect on cell viability, since the growth rates were reduced in both cell lines (Figure 1C).


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Figure 5: Quantification of aconitase and isocitrate dehydrogenase activities. (A) The activities isocitrate dehydrogenase and (B) of aconitase were quantified in HEK293T wild type (WT) and heterozygous (+/-) or homozygous (-/-) MOCS3 knockout cells after cells lysis in NP-40 buffer. The data are mean values from three independent measurements (± S.D.) The Pvalues were calculated using an unpaired t-test (n = 3) (ns P>0.05, * P≤0.05, *** P≤0.001, **** P≤0.0001). (C) and (D) Immunoblotting with antibodies raised against (C) isocitrate dehydrogenase (ICDH) or (D) mitochondrial aconitase (Aco). The cell lines were lysed with NP40 buffer and separated by 12% SDS-PAGE. The proteins were blotted on a PVDF membrane and visualized with antibodies against ICDH and mitochondrial aconitase using a POD-labelled secondary antibody. Actin was used as a loading control. Localization studies revealed an accumulation of NFS1 at the Centrosome. In previous studies, we showed that the L-cysteine desulfurase NFS1 interacts with MOCS3 in the cytosol for sulfur transfer to both Moco biosynthesis and thiomodification of mcm5s2U modified nucleosides in tRNA.25,

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A role of NFS1 in cytosolic Fe-S cluster assembly is, however,

controversial. One view is that CIA machinery is independent of cytosolic NFS1, while another model is that cytosolic NFS1 is the sulfur donor for cytosolic Fe-S cluster assembly, which is independent of the mitochondrial version of NFS1. Since the cytosolic levels of NFS1 are very low, there might be a higher demand of cytosolic NFS1 under conditions when NFS1 is required for


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Moco biosynthesis and tRNA tholation. To analyse whether MOCS3 influences the cellular localization of NFS1 in the cytosol, we determined the localization of NFS1 in HEK293T wildtype cells and MOCS3 (-/-) knockout cells by indirect immunofluorescence imaging using an antibody derived against NFS1. Indirect immunofluorescence determined a localization of NFS1 both in the mitochondria and in the cytosol in both cell lines (Figure 6). Unexpectedly, in these images NFS1 was also found at the centrosome as revealed by its strong co-localization with the centrosomal marker protein γ-tubulin (Figure 6, enlarged images). This localization was not only detected in HEK293T cells but was also verified in HeLa cells (Supplementary Figure S3). To obtain a better resolution of the localization of NFS1 at the centrosome, z-stack images were recorded with a wide-field microscope and deconvolved (Figure 7A). The results reveal a clear colocalization of NFS1 with γ-tubulin when recording a z-stack cut through the centre of the centrioles, with an accumulation of both proteins to one end of the centrioles (Figure 7B). To verify this novel localization of NFS1, we isolated centrosomes from HEK293T cells via a sucrose density gradient and identified the presence of NFS1 using an -NFS1 antibody. Different fractions were collected from the bottom to the top of the gradient. The centrosomes showed the highest centrosome concentration in fraction 12, which corresponded to approximately 60% sucrose. This was verified by the presence of the centrosomal proteins γ-tubulin and Cdk5Rap2. NFS1 was also detected in this centrosome fraction (Figure 8A). In order to exclude any cytoplasmic or mitochondrial contaminations in the centrosomal fraction, the proteins were additionally transferred onto coverslips for confocal microscopy. This technique allows the particular visualisation of the centrosomes and additionally allows for overlay of images of the centrosomes and the protein of interest. The results in Figure 8B show that NFS1 clearly colocalizes with γ-tubulin. The same co-localization was obtained when using the centriolar tubulin


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marker GT335 as control recognizing glutamylated tubulin, thereby verifying the novel localization of NFS1 at the centrosome.

Figure 6: Indirect immunofluorescence of NFS1 in HEK293T Cells. NFS1 was analyzed in HEK293T cells for subcellular localization and colocalization by confocal fluorescence microscopy. Cells were fixed onto cover slips and analyzed under a LSM710 confocal microscope. NFS1 was labelled with antibodies (green). DAPI (blue) was used to stain the nucleus and γ-tubulin (red) as a centrosomal marker. The insets show a zoomed in part of the centrosome to better visualize the co-localization.


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Figure 7: NFS1 Accumulates at the Tips of the Centrioles. A) HeLa cells were stained with antibodies against NFS1 (green) and compared to γ-tubulin (red). Z-stack images of the centrosomes were obtained using widefield microscopy. The qualities of the images were improved using deconvolution. B) The plot profiles represent the cross-sections of the centrosomes. C) Schematic representation of the predicted centriole orientation showing NFS1 (green) and γ-tubulin (red). Yellow shows the co-localization of NFS1 and γ-tubulin.


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Figure 8: Localization studies of NFS1 at fractionated centrosomes. (A) The centrosomes from Hek293T cells were isolated via a sucrose gradient. Fractions of the 4060% sucrose layer were collected. Proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane. Immunoblotting with antibodies raised against centrosomal proteins (γ-tubulin and Cdk5Rap2), a nuclear envelope protein (LaminB1), and NFS1 was performed. Proteins were visualized with a POD-labelled secondary antibody. (B) The centrosomes from fraction 12 were centrifuged onto cover slips and observed under the LSM710 confocal microscope. γ-tubulin (red) was used as a centrosomal marker and its localization was compared to that of NFS1 (green). Zoomin Images were made comparing the localization of NFS1 (green) to γ-tubulin and GT335 (red).


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DISCUSSION So far, the majority of patients suffering from Moco deficiency were described with mutations in MOCS1, MOCS2, or GEPH.8 These patients generally were seriously affected in the neonatal period with severe symptoms including seizures, convulsions, skeletal changes, and dysmorphic features, often leading to death in early childhood.4 Only recently the first patient with a genetic defect in MOCS3 has been described, which exhibited milder symptoms and never experienced epileptic seizures.12 The mutation revealed to result in an A257T amino acid exchange in the Nterminal MoeB-like domain of MOCS3. Based on the mild symptoms of the patient, likely the A257T amino acid exchange not completely affected the adenylation of either MOCS2A or URM1, especially since normal sulfite oxidase activities were detected in fibroblasts. In this report, we were the first to construct a complete MOCS3 knockout in human HEK293T cell lines to analyze the role of MOCS3 for Moco biosynthesis, tRNA thiolation and overall cell viability. We show that in contrast to the report of the human patient, the homozygous MOCS3 knockout abolished sulfite oxidase activity almost completely based on the absence of Moco in these cells. Simultaneously, cPMP accumulated and in addition, no mcm5s2U thio-modified nucleosides in tRNA were identified. We therefore expect that the symptoms of a knockout patient in MOCS3 should be identical to the symptoms of patients in MOCS1, MOCS2 or GEPH, based on the inactivity of sulfite oxidase. Conclusively, in the described patient with an A257T amino exchange in MOCS3, the MOCS3 protein is expected to have residual activities which then result in sufficient Moco production to guarantee for sulfite oxidase activity. Unfortunately, the levels of Moco or mcm5s2U thiomodifications in tRNA were not quantified in this patient. In our study, we show that in the absence of Moco, also the sulfite oxidase protein backbone was not identified by immunodetection. This result is in agreement with a previous report investigating


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the maturation of sulfite oxidase in mitochondria.58 In this report it has been suggested that the maturation of sulfite oxidase is Moco dependent. In the absence of Moco, most of the unmatured apo-protein was found to be present in the cytosol. Since in our study, no sulfite oxidase was detected, likely the apo-form of sulfite oxidase is immediately degraded in the cytosol. Other molybdoenzymes like xanthine dehydrogenase or aldehyde oxidase were not tested in our study, since the HEK293T cells contain only low amounts of these enzymes and the activities were below the limit of detection. The low activities are further reflected in the low Moco levels of these cells. Cells that would produce higher amounts of Moco and active molybdoenzymes are liver cells, while in kidney cells only low amounts of Moco are produced. We therefore explain the low levels of Moco and sulfite oxidase activity in this study by the fact that HEK293T cells were used. We used HEK293T cells in our study since these cell lines can be better handled for cellular localization and transfection experiments. This also might be the reason why we only detected a low amount of cPMP accumulation in the MOCS3 (-/-) cells. Patients with a type B molybdenum cofactor deficiency (with mutations in the MOCS2A or MOCS2B genes) were reported to excrete cPMP in the urine, which can be used to diagnose the disease.59 In humans, the main organ is the liver which produces most of the cellular cPMP. We also identified that the absence of MOCS3 affected the overall cell viability of the HEK293T cells, since the knockout cells reached after three days of growth only half of the cell mass. This retarded cell growth might be based on an impaired aconitase activity, the activity of which was also 50% reduced in the MOCS3 knockout cells. In contrast, isocitrate dehydrogenase was not affected in these cells, an enzyme that does not require any additional cofactors for activity. Whether the reduced aconitase activity is based on reduced levels of Fe-S clusters in the MOCS3 knockout cells was not determined, additional detailed analyses would be required to analyze the


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cellular Fe-S cluster levels in the mitochondria and cytosol separately. However, while we could not directly explain the effect of the MOCS3 knockout on aconitase activity, we additionally investigated the localization of NFS1 as interaction partner of MOCS3 in the cytosol. Previous studies by our group identified NFS1 as a sulfur donor for MOCS3 in the cytosol.25, 26 A recent study confirmed the interaction of NFS1 and MOCS3 in the cytosol, identifying MOCS3 and URM1 as interaction partners of NFS1 by specific co-immunoprecipitation of endogenous cytosolic NFS1 in HEK293T cells.50 Interestingly, our localization studies identified NFS1 at the centrosome. This localization, however, was independent of MOCS3. Therefore, MOCS3 does not seem to impact the cytosolic localization of NFS1. So far, NFS1 was described to be mainly localized to the mitochondria and the nucleus. In mitochondria, the main role of NFS1 has been described to form a complex with ISD11, ISCU and frataxin for Fe-S cluster biosynthesis. Recent crystal structures of the quaternary complex also identified the acyl carrier protein to be present in this complex under certain conditions.60, 61 In the nucleus, the NFS1/ISD11 complex is also predicted to be involved in Fe-S cluster biosynthesis for nuclear proteins. The identification of NFS1 at the centrosome is another proof for the cytosolic localization of NFS1. A report investigating the centrosomal proteins in Drosophila already identified NFS1 (referred to as CG12264 in this publication) as a centrosomal protein.62 This localization was verified for human cell lines in our study. The role of NFS1 at the centrosome still remains to be identified In a recent report by Kim et al. (2018)50, NFS1 was co-immunoprecipitated with the centrosomal protein NUBP2. NUBP2 itself interacts with the kinesin-like protein HSET (KIFC5A in mice) and is an important factor of the cytosolic iron-sulfur cluster machinery (CIA).63, 64 For the CIA pathway, NUBP2 forms a larger complex with Cfd1-Nbp35 acting as a scaffold for [4Fe-


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Biochemistry

4S] cluster assembly and insertion.44 Further, the chromokinesin KIF4A has been shown recently to bind a Fe-S cluster through its conserved cysteine-rich domain.65 A colocalization of the CIA complex with components of the mitotic machinery have been demonstrated in this report, with the suggested role to facilitate the transfer of Fe-S clusters to KIF4A. The authors speculated that the lack of Fe-S clusters in KIF4A upon downregulation of the CIA targeting complex might contribute to mitotic defects. Conclusively, not only the CIA complex might be involved in inserting Fe-S clusters into KIF4A, since NFS1 localized at the centrosomes might directly be involved in this process. Thus, the role of NFS1 at the centrosome might be to insert Fe-S clusters into centrosomal and/or chromosomeassociated proteins, thereby influencing cell division. The exact role of NFS1 at the centrosome in addition to its interaction partners, however, need to be investigated in further detail in future studies.


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AUTHOR INFORMATION Corresponding Author *To whom correspondence should be addressed: Silke Leimkühler, Institute of Biochemistry and Biology, Department of Molecular Enzymology, University of Potsdam, Karl-Liebknecht-Str. 2425, 14476 Potsdam, Germany, Telephone: +49-331-977-5603; Fax: +49-331 977-5128; E-mail: [email protected] Funding The research leading to these results has received funding from the International Max Planck Research School on Multiscale Biosystems to Y.N.. This work was also supported by the Deutsche Forschungsgemeinschaft grant LE1171/9-2 to SL. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Prof. Dr. Klemens Rottner (University of Braunschweig, Germany) for CRISPR/Cas9 vectors and protocols, Prof. Dr. Angela Kaindl (Charité, Berlin, Germany) for anti-CDK5RAP2 antibodies, Prof. Dr. Ingrid Hofmann (DKFZ, Heidelberg, Germany), Prof. Dr. Oliver Gruss (University of Bonn, Germany) and Prof. Dr. Carsten Janke (Institut Curie, Paris, France) for GT335 antibodies. ABBREVIATIONS


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frataxin (FXN) gephyrin (GPHN) molybdenum cofactor (Moco) cyclic pyranopterin monophosphate (cPMP) molybdopterin (MPT) succinate dehydrogenase (SDH) molybdenum cofactor synthesis protein 3 (MOCS3)

SUPPORTING INFORMATION AVAILABLE Supplementary Figures S1 to S3. Figure S1: Identified base pair exchanges in the heterozygous (+/-) MOCS3 knockout cell line. Figure S2: Identified base pair exchanges in the homozygous (-/-) MOCS3 knockout cell line. Figure S3: Indirect immunofluorescence of NFS1 in HeLa Cells.

PROTEIN ACCESSION IDs: MOCS3: O95396; NFS1: Q9Y697


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REFERENCES [1] Rajagopalan, K. V., Johnson, J. L., and Hainline, B. E. (1982) The pterin of the molybdenum cofactor, Fed. Proc. 41, 2608-2612. [2] Hille, R., Hall, J., and Basu, P. (2014) The mononuclear molybdenum enzymes, Chemical reviews 114, 3963-4038. [3] Mendel, R. R., and Kruse, T. (2012) Cell biology of molybdenum in plants and humans, Biochim Biophys Acta 1823, 1568-1579. [4] Duran, M., Beemer, F. A., van der Heiden, C., Korteland, J., de Bree, P. K., Brink, M., Wadman, S. K., and Lombeck, I. (1978) Combined deficiency of xanthine oxidase and sulphite oxidase: a defect of molybdenum metabolism or transport?, J. Inher. Metab. Dis. 1, 175-178. [5] Duran, M., de Bree, P. K., de Klerk, J. B. C., Dorland, L., and Berger, R. (1996) Molybdenum cofactor deficiency: clinical presentation and laboratory diagnosis, Int. Pediatr. 11, 334338. [6] Schwarz, G. (2005) Molybdenum cofactor biosynthesis and deficiency, Cell Mol Life Sci 62, 2792-2810. [7] Johnson, J. L., and Duran, M. (2001) Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency, In The Metabolic and Molecular Bases of Inherited Disease, 8th edition (Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Childs, B., and Vogelstein, B., Eds.), pp 3163-3177, McGraw-Hill, New York. [8] Reiss, J., and Hahnewald, R. (2011) Molybdenum cofactor deficiency: Mutations in GPHN, MOCS1, and MOCS2, Hum Mutat 32, 10-18.


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For table of contents use only

MOCS3 has a role in multiple cellular pathways in human cells


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Analysis of the cellular roles of MOCS3 identifies a MOCS3

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