Dual role of the molybdenum cofactor biosynthesis protein MOCS3 in tRNA thiolation and molybdenum cofactor biosynthesis in humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.     

Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis

The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, M?ssbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe-4S](2+,+) cluster is ligated by an N-terminal CX(3)CX(2)C motif as is the case with all other AdoMet-dependent radical enzymes investigated thus far. A C-terminal CX(2)CX(13)C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe-4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S](2+) clusters. The N-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe-2S](2+) cluster intermediate, and the C-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [3Fe-4S](0) cluster intermediate.     

A novel role for human Nfs1 in the cytoplasm: Nfs1 acts as a sulfur donor for MOCS3, a protein involved in molybdenum cofactor biosynthesis

The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an l-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human l-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from l-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco.     

Structure-activity relationships by mass spectrometry: identification of novel MMP-3 inhibitors

A novel class of nonpeptide inhibitors of stromelysin (MMP-3) has been discovered with the use of mass spectrometry. The method relies on the development of structure-activity relationships by mass spectrometry (SAR by MS) and utilizes information derived from the binding of known inhibitors to identify novel inhibitors of a target protein with a minimum of synthetic effort. Noncovalent complexes of known inhibitors with a target protein are analyzed; these inhibitors are deconstructed into sets of fragments which compete for common or overlapping binding sites on the target protein. The binding of each fragment set can be studied independently. With the use of competition studies, novel members of each fragment set are identified from compound libraries that bind to the same site on the target protein. A novel inhibitor of the target protein was then constructed by chemically linking a combination of members of each fragment set in a manner guided by the proximity and orientation of the fragments derived from the known inhibitors. In the case of stromelysin, a novel inhibitor composed of favorably linked fragments was observed to form a 1:1 complex with stromelysin. Compounds that were not linked appropriately formed higher order complexes with stoichiometries of 2:1 or greater. These linked molecules were subsequently assessed for their ability to block stromelysin function in a chromogenic substrate assay.     

Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3. Major differences in substrate specificity between eukaryotic and bacterial homologs

Sequence alignments of human molybdopterin synthase sulfurase, MOCS3, showed that the N-terminal domain is homologous to Escherichia coli MoeB, whereas the C-terminal domain is homologous to rhodanese-like proteins. Previous studies showed that the activity of the separately purified rhodanese-like domain of MOCS3 displayed 1000-fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. When the six amino acid active site loop of MOCS3 rhodanese-like domain was exchanged with the loop found in bovine rhodanese, thiosulfate:cyanide sulfurtransferase activity was increased 165-fold. Site-directed mutagenesis of each individual residue of the active site loop of the MOCS3 rhodanese-like domain showed that the charge of the last amino acid determines thiosulfate sulfurtransferase activity. Replacing Asp417 by threonine resulted in 90-fold increased activity, whereas replacing it by arginine increased the activity 470-fold. Using a fully defined in vitro system containing precursor Z, MOCS2A, E. coli MoaE, E. coli MoeB, Mg-ATP, MOCS3 rhodanese-like domain, and thiosulfate, it was shown that sulfur transfer to MOCS2A was also affected by the alterations, but not as drastically. Our studies revealed that in humans and most eukaryotes thiosulfate is not the physiologic sulfur donor for MOCS3, whereas in bacterial homologs, which have an arginine at the last position of the active site loop, thiosulfate can be used as a sulfur source for molybdenum cofactor biosynthesis. The phylogenetic analysis of MoeB homologs showed that eukaryotic homologs are of bacterial origin. Furthermore, it could be shown that an MoeB homolog named MoeZ, where the dual CXXC zinc-binding motif of the MoeB domain is not present, arose independently several times during evolution.     

Ten novel mutations in the molybdenum cofactor genes MOCS1 and MOCS2 and in vitro characterization of a MOCS2 mutation that abolishes the binding ability of molybdopterin synthase

Molybdenum cofactor deficiency (MIM#252150) is a severe autosomal-recessive disorder with a devastating outcome. The cofactor is the product of a complex biosynthetic pathway involving four different genes (MOCS1, MOCS2, MOCS3 and GEPH). This disorder is caused almost exclusively by mutations in the MOCS1 or MOCS2 genes. Mutations affecting this biosynthetic pathway result in a lethal phenotype manifested by progressive neurological damage via the inactivation of the molybdenum cofactor-dependent enzyme, sulphite oxidase. Here we describe a total of ten novel disease-causing mutations in the MOCS1 and MOCS2 genes. Nine out of these ten mutations were classified as pathogenic in nature, since they create a stop codon, affect constitutive splice site positions, or change strictly conserved motifs. The tenth mutation abolishes the stop codon of the MOCS2B gene, thus elongating the corresponding protein. The mutation was expressed in vitro and was found to abolish the binding affinities of the large subunit of molybdopterin synthase (MOCS2B) for both precursor Z and the small subunit of molybdopterin synthase (MOCS2A).     

Blood group A glycolipid antigen biosynthesis: discrimination between biosynthesized and enzyme preparation derived blood group A antigen by mass spectrometry

A monofucosyl type 1 chain blood group A hexaglycosylceramide was biosynthesized in solution using the type 1 chain blood group H pentaglycosylceramide as precursor, a crude microsomal fraction prepared from the mucosa scraping of a blood group A pig small intestine as enzyme source, and uridine diphosphate-N-acetyl-(1-14C)galactosamine as sugar donor. The radioactive product was enriched using reversed-phase column chromatography and silica gel HPLC. The peak, as detected by a beta-flow scintillation counter, was collected, permethylated, and analyzed by mass spectrometry. Carbohydrate sequence ions were found, indicating the presence of both the biosynthesized and a native, non-14C-containing blood group A hexaglycosylceramide. The blood group A pig small intestinal mucosa used as the enzyme source contain blood group A hexaglycosylceramide as the predominant glycolipid. Therefore, it is concluded that the nonradioactive blood group A hexaglycosylceramide found after the biosynthesis is derived from the enzyme preparation.     

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.     

1-(3-Aminopropyl)-3-butylimidazolium bromide for carboxyl group derivatization: potential applications in high sensitivity peptide identification by mass spectrometry

The cationic reagent 1-(3-aminopropyl)-3-butylimidazolium bromide(BAPI) was exploited for the derivatization of carboxyl groups on peptides.Nearly 100% derivatization efficiency was achieved with the synthetic peptide RVYVHPI(RI-7).Furthermore,the peptide derivative was stable in a 0.1% TFA/water solution or a 0.1%(v/v) TFA/acetonitrile/water solution for at least one week.The effect of BAPI derivatization on the ionization of the peptide RI-7 was further investigated,and the detection sensitivity was improved >42-fold via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS),thus outperforming the commercial piperazine derivatization approach.Moreover,the charge states of the peptide were largely increased via BAPI derivatization by electrospray ionization(ESI) MS.The results indicate the potential merits of BAPI derivatization for high sensitivity peptide analysis by MS.     

Direct identification of a G protein ubiquitination site by mass spectrometry

Covalent attachment of ubiquitin is well-known to target proteins for degradation. Here, mass spectrometry was used to identify the site of ubiquitination in Gpa1, the G protein alpha subunit in yeast Saccharomyces cerevisiae. The modified residue is located at Lys165 within the alpha-helical domain of Galpha, a region of unknown function. Substitution of Lys165 with Arg (Gpa1(K165R)) results in a substantial decrease in ubiquitination. In addition, yeast expressing the Gpa1(K165R) mutant are moderately resistant to pheromone in growth inhibition assays-a phenotype consistent with enhanced Galpha signaling activity. These findings indicate that the alpha-helical domain may serve to regulate the turnover of Gpa1.     

Strain identification of commercial influenza vaccines by mass spectrometry

Current influenza vaccine manufacturing and testing timelines require that the constituent hemagglutinin (HA) and neuraminidase (NA) strains be selected each year approximately 10 months before the vaccine becomes available. The threat of a pandemic influenza outbreak requires that more rapid testing methods be found. We have developed a specialized on-filter sample preparation method that uses both trypsin and chymotrypsin to enzymatically digest peptide-N-glycosidase F (PNGase F)-deglycosylated proteins in vaccines. In tandem with replicate liquid chromatography-mass spectrometry (LC-MS) analyses, this approach yields sufficient protein sequencing data (>85% sequence coverage on average) for strain identification of HA and NA components. This has allowed the confirmation, and in some cases the correction, of the identity of the influenza strains in recent commercial vaccines as well as the correction of some ambiguous HA sequence annotations in available databases. This method also allows the identification of low-level contaminant egg proteins produced during the manufacturing process.     

Rapid identification of DNA-binding proteins by mass spectrometry.

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.     

Protein identification strategies in MALDI imaging mass spectrometry: a brief review

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Confident identification of 3-nitrotyrosine modifications in mass spectral data across multiple mass spectrometry platforms

3-nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometry-based proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NT-containing peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LC-MS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NT-containing peptides with significant overlap in the peptides detected by both systems. We investigated the LC-MS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NT-containing peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification.