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Characterization of PdCP1, a Serine Carboxypeptidase from P. destructans, the Causal Agent of White-nose Syndrome

ABSTRACT: Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome, a disease afflicting multiple species of North American bats. P. destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were discovered, including a putative serine peptidase, PdCP1 that was unique to the P. destructans secretome. This enzyme was expressed in yeast and the substrate preference was discovered using a multiplexed-substrate profiling method based on enzymatic degradation of a sequence-diverse synthetic peptide library. The cleaved products were identified by mass spectrometry. In general, the peptide substrates were sequentially truncated from the carboxyl terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located in the penultimate position were rapidly cleaved and therefore a fluorescent substrate containing arginine was used to biochemically characterize the enzyme and screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be potent inhibitors of PdCP1 activity.

INSTRUMENT(S): LTQ Orbitrap Velos, LTQ Orbitrap XL, Q Exactive

ORGANISM(S): Pseudogymnoascus Destructans Pseudogymnoascus Destructans (ncbitaxon:655981)

SUBMITTER: Anthony O'Donoghue Richard J. Bennett

PROVIDER: MSV000082301 | MassIVE | Sun Apr 22 22:59:00 BST 2018

REPOSITORIES: MassIVE

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Project description:Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (~0.1% of total arginine), Rme2a was highly abundant (~1.1%), and Rme2s was intermediate (~0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

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Characterization of PdCP1, a Serine Carboxypeptidase from P. destructans, the Causal Agent of White-nose Syndrome

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