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Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC² 155 by proteogenomic analysis.

ABSTRACT: Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary b/y ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Trypsin and Ac-LysargiNase, were used in Mycolicibacterium smegmatis MC² 155 for proteogenomic analysis. The mirror proteases accurately identified 368 novel peptides, exhibiting 75-80% b and y ion coverages against 65-68% y or b ion coverages of Ac-Trypsin (38.9% b and 68.3% y) or Ac-LysargiNase (65.5% b and 39.6% y) as annotated peptides from M. smegmatis MC² 155. The complementary b and y ion series largely increased the reliability of overlapped sequences derived from novel peptides. Among these novel peptides, 311 peptides were annotated in other public M. smegmatis strains, and 57 novel peptides with more continuous b and y pairs were obtained for further analysis after spectral quality assessment. This enabled mirror proteases to successfully correct six annotated proteins' N-termini and detect 17 new coding open reading frames (ORFs). We believe that mirror proteases will be an effective strategy for novel peptide detection in both prokaryotic and eukaryotic proteogenomics.

SUBMITTER: Jiang S

PROVIDER: S-EPMC9597629 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC2 155 by proteogenomic analysis.

Jiang Songhao S Shi Jiahui J Li Yanchang Y Zhang Zhenpeng Z Chang Lei L Wang Guibin G Wu Wenhui W Yu Liyan L Dai Erhei E Zhang Lixia L Lyu Zhitang Z Xu Ping P Zhang Yao Y

Frontiers in microbiology 20221012

Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary b/y ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Tryps ...[more]

PMID: 36312923

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Project description:BackgroundPregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones.ResultsBased on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses.ConclusionThe use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Project description:Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing posttranslational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of posttranslational phosphoarginine modifications in Mycolicibacterium smegmatis, a nonpathogenic surrogate of M. tuberculosis. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identified arginine phosphosites on several functionally diverse targets within the M. smegmatis proteome. Interestingly, phosphoarginine modifications are not upregulated by heat stress, suggesting divergent roles in mycobacteria and Bacillus. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species. IMPORTANCE Mycobacteria that cause tuberculosis infections employ proteolytic pathways that modulate cellular behavior by destroying specific proteins in a highly regulated manner. Some proteolytic enzymes have emerged as novel antibacterial targets against drug-resistant tuberculosis infections. However, we have only a limited understanding of how these enzymes function in the cell and how they select proteins for destruction. Some proteolytic enzymes are capable of recognizing proteins that carry an unusual chemical modification, arginine phosphorylation. Here, we confirm the existence of arginine phosphorylation in mycobacterial proteins. Our work expands our understanding of a promising drug target in an important global pathogen.

Project description:The prevalence of drug-resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new drug targets in this globally important pathogen. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize compounds that impinge on ClpC1 activity are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a coimmunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a surrogate for M. tuberculosis. We identify a diverse panel of interaction partners, many of which coimmunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis establishes MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires exposure of its N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs on substrates. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics to help address the challenge of M. tuberculosis drug resistance. IMPORTANCE Drug-resistant tuberculosis infections are a major challenge to global public health. Much effort has been invested in identifying new drug targets in the causative pathogen, Mycobacterium tuberculosis. One such target is the ClpC1 unfoldase. Compounds have been identified that kill M. tuberculosis by disrupting ClpC1 activity, yet the physiological function of ClpC1 in cells has remained poorly defined. Here, we identify interaction partners of ClpC1 in a model mycobacterium. By building a broader understanding of the role of this prospective drug target, we can more effectively develop compounds that inhibit its essential cellular activities.

Dataset Information

Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC² 155 by proteogenomic analysis.

Publications

Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in <i>Mycolicibacterium smegmatis</i> MC<sup>2</sup> 155 by proteogenomic analysis.

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