Hem25p is required for mitochondrial IPP transport in fungi.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor composed of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for haem biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p failed to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enabled robust IPP uptake and incorporation into the CoQ biosynthetic pathway. HEM25 orthologues from diverse fungi, but not from metazoans, were able to rescue hem25∆ CoQ deficiency. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in fungi.

Hem25p is a mitochondrial IPP transporter.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor comprised of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing, and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for heme biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p fail to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enables robust IPP uptake demonstrating that Hem25p is sufficient for IPP transport. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in yeast.

Nitric oxide-driven modifications of lipoic arm inhibit α-ketoacid dehydrogenases.

Pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC), which belong to the mitochondrial α-ketoacid dehydrogenase family, play crucial roles in cellular metabolism. These multi-subunit enzyme complexes use lipoic arms covalently attached to their E2 subunits to transfer an acyl group to coenzyme A (CoA). Here, we report a novel mechanism capable of substantially inhibiting PDHC and OGDC: reactive nitrogen species (RNS) can covalently modify the thiols on their lipoic arms, generating a series of adducts that block catalytic activity. S-Nitroso-CoA, a product between RNS and the E2 subunit's natural substrate, CoA, can efficiently deliver these modifications onto the lipoic arm. We found RNS-mediated inhibition of PDHC and OGDC occurs during classical macrophage activation, driving significant rewiring of cellular metabolism over time. This work provides a new mechanistic link between RNS and mitochondrial metabolism with potential relevance for numerous physiological and pathological conditions in which RNS accumulate.

Small-molecule inhibition of the archetypal UbiB protein COQ8.

Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

Mass Spectrometry-Cleavable Protein N-Terminal Tagging Strategy for System-Level Protease Activity Profiling.

Proteolysis is one of the most important protein post-translational modifications (PTMs) that influences the functions, activities, and structures of nearly all proteins during their lifetime. To facilitate the targeted identification of low-abundant proteolytic products, we devised a strategy incorporating a novel biotinylated reagent PFP (pentafluorophenyl)-Rink-biotin to specifically target, enrich and identify proteolytic N-termini. Within the PFP-Rink-biotin reagent, a mass spectrometry (MS)-cleavable feature was designed to assist in the unambiguous confirmation of the enriched proteolytic N-termini. The proof-of-concept study was performed with multiple standard proteins whose N-termini were successfully modified, enriched and identified by a signature ion (SI) in the MS/MS fragmentation, along with the determination of N-terminal peptide sequences by multistage tandem MS of the complementary fragment generated after the cleavage of MS-cleavable bond. For large-scale application, the enrichment and identification of protein N-termini from Escherichia coli cells were demonstrated, facilitated by an in-house developed NTermFinder bioinformatics workflow. We believe this approach will be beneficial in improving the confidence of identifying proteolytic substrates in a native cellular environment.

Dual-Stage Neutral Loss Tandem Mass Spectrometric Strategy for Confident Identification of Protein Prenylation.

Protein prenylation is an important post-translational modification that regulates protein interactions, localizations, and signaling pathways in normal functioning of eukaryotic cells. It is also a critical step in the oncogenic developments of various cancers. Direct identification of native protein prenylation by mass spectrometry (MS) has been challenging due to high hydrophobicity and the lack of an efficient enrichment technique. Prior MS studies of prenylation revealed that prenyl peptides readily generate high-intensity fragments after neutral loss of the prenyl group (R group), and more recent investigation of oxidized prenyl peptides discovered more consistent neutral loss of the oxidized prenyl group (RSOH group). Here, a dual-stage neutral loss MS3 (DS-NLMS3)-based strategy is therefore developed by combining both gas-phase cleavable properties of the prenyl thioether bond and mono-oxidized thioether to improve the large-scale identification of prenylation. Both neutral losses can individually and distinctively confirm the prenylation type in MS2 and the sequence of the prenyl peptide upon targeted MS3 fragmentation. This dual-faceted NLMS3 strategy significantly improves the confidence in the identification of protein prenylation from large-scale samples, which enables the unambiguous identification of prenylated sites of the spiked low-abundance farnesyl peptide and native prenyl proteins from mouse macrophage cells, even without prior enrichment during sample preparation. The ease of incorporating this strategy into the prenylation study workflow and minimum disruption to the biological lipidome are advantageous for unraveling unknown native protein prenylation and further developments in profiling and quantifying prenylome.

Affinity and chemical enrichment strategies for mapping low-abundance protein modifications and protein-interaction networks.

Protein post-translational modifications and protein interactions are the central research areas in mass-spectrometry-based proteomics. Protein post-translational modifications affect protein structures, stabilities, activities, and all cellular processes are achieved by interactions among proteins and protein complexes. With the continuing advancements of mass spectrometry instrumentations of better sensitivity, speed, and performance, selective enrichment of modifications/interactions of interest from complex cellular matrices during the sample preparation has become the overwhelming bottleneck in the proteomics workflow. Therefore, many strategies have been developed to address this issue by targeting specific modifications/interactions based on their physical properties or chemical reactivities, but only a few have been successfully applied for systematic proteome-wide study. In this review, we summarized the highlights of recent developments in the affinity enrichment methods focusing mainly on low stoichiometric protein lipidations. Besides, to identify potential glyoxal modified arginines, a small part was added for profiling reactive arginine sites using an enrichment reagent. A detailed section was provided for the enrichment of protein interactions by affinity purification and chemical cross-linking, to shed light on the potentials of different enrichment strategies, along with the unique challenges in investigating individual protein post-translational modification or protein interaction network.

Improved in-solution trypsin digestion method for methanol-chloroform precipitated cellular proteomics sample.

Methanol-chloroform based protein precipitation is an essential step in many liquid chromatography-tandem mass spectrometry-based cellular proteomics applications. However, re-solubilization of the total protein precipitate is difficult using regular in-solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re-solubilization of membrane fractions. In this study, we demonstrated an application combining methanol-chloroform based protein precipitations and deoxycholic acid assisted re-solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry-based bottom-up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate. Detailed in-solution trypsin digestion studies were presented on methanol-chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid-treated samples compared to other established methods. Furthermore, liquid chromatography-tandem mass spectrometry analysis of an equal amount of proteins from methanol-chloroform precipitated, and methanol-chloroform/deoxycholic acid-treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used.

Evaluation of Different Stationary Phases in the Separation of Inter-Cross-Linked Peptides.

Chemical cross-linking coupled with mass spectrometry (MS) is becoming a routinely and widely used technique for depicting and constructing protein structures and protein interaction networks. One major challenge for cross-linking/MS is the determination of informative low-abundant inter-cross-linked products, generated within a sample of high complexity. A C18 stationary phase is the conventional means for reversed-phase (RP) separation of inter-cross-linked peptides. Various RP stationary phases, which provide different selectivities and retentions, have been developed as alternatives to C18 stationary phases. In this study, two phenyl-based columns, biphenyl and fluorophenyl, were investigated and compared with a C18 phase for separating BS3 (bis(sulfosuccinimidyl)suberate) cross-linked bovine serum albumin (BSA) and myoglobin by bottom-up proteomics. Fractions from the three columns were collected and analyzed in a linear ion trap (LIT) mass spectrometer for improving detection of low abundant inter-cross-linked peptides. Among these three columns, the fluorophenyl column provides additional ion-exchange interaction and exhibits unique retention in separating the cross-linked peptides. The fractioned data was analyzed in pLink, showing the fluorophenyl column consistently obtained more inter-cross-linked peptide identifications than both C18 and biphenyl columns. For the BSA cross-linked sample, the identified inter-cross-linked peptide numbers of the fluorophenyl to C18 column are 136 to 102 in "low confident" results and 11 to 6 in "high confident" results. The fluorophenyl column could potentially be a better alternative for targeting the low stoichiometric inter-cross-linked peptides.