Cryptococcus neoformans Prp8 Intein: An In Vivo Target-Based Drug Screening System in Saccharomyces cerevisiae to Identify Protein Splicing Inhibitors and Explore Its Dynamics
Abstract
:1. Introduction
2. Materials and Methods
2.1. System Design and Work
2.2. Cloning, Site-Directed Mutagenesis and S. cerevisiae Transformation
2.2.1. DNA and RNA Manipulation
2.2.2. Site-Directed Mutagenesis by Inverse PCR
2.2.3. S. cerevisiae Manipulation
2.3. Protein Analysis
2.4. Bioinformatic Analysis
2.5. Cisplatin Assay
3. Results
3.1. Yeast Growth Pattern (Temperature Sensitivity)
3.2. Assessment of CnePrp8i Presence at mRNA Level
3.3. Evaluation of CnePrp8i Splicing by Western Blot
3.4. Test of Cisplatin in the Proposed In Vivo System
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
5-FOA | 5-Fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate) |
AIDS | Acquired immunodeficiency syndrome |
CgaPrp8i | Cryptococcus gattii Prp8 intein |
CnePrp8i | Cryptococcus neoformans Prp8 intein |
eiF5a | Eukaryotic translation initiation factor 5A |
EUCAST | European Committee on Antimicrobial Susceptibility Testing |
HE | Homing endonuclease |
HIV | Human immunodeficiency virus |
HRP | Horseradish peroxidase |
IC50 | Half-maximal inhibitory concentration |
iPAS | Intein-mediated protein alternative splicing |
MIC | Half-maximal inhibitory concentration |
MS | Mass spectrometry |
MtuRecA | Mycobacterium tuberculosis recombinase A protein |
MtuRecAi | Mycobacterium tuberculosis recombinase A intein |
OMP | Orotidine-5′-phosphate |
Prp8 | Pre-mRNA-splicing factor 8 |
RadA | Archaeal RecA protein homolog |
RecA | Recombinase A protein |
RT | Reverse transcriptase |
SC | Synthetic complete (medium) |
ssDNA | Single-stranded DNA |
SufB | Fe-S cluster assembly protein |
UMP | Uridine monophosphate |
URA3 | Orotidine 5-phosphate decarboxylase |
YPD | Yeast extract–peptone–dextrose (medium) |
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Fernandes, J.A.L.; Zatti, M.d.S.; Arantes, T.D.; Souza, M.F.B.d.; Santoni, M.M.; Rossi, D.; Zanelli, C.F.; Liu, X.-Q.; Bagagli, E.; Theodoro, R.C. Cryptococcus neoformans Prp8 Intein: An In Vivo Target-Based Drug Screening System in Saccharomyces cerevisiae to Identify Protein Splicing Inhibitors and Explore Its Dynamics. J. Fungi 2022, 8, 846. https://doi.org/10.3390/jof8080846
Fernandes JAL, Zatti MdS, Arantes TD, Souza MFBd, Santoni MM, Rossi D, Zanelli CF, Liu X-Q, Bagagli E, Theodoro RC. Cryptococcus neoformans Prp8 Intein: An In Vivo Target-Based Drug Screening System in Saccharomyces cerevisiae to Identify Protein Splicing Inhibitors and Explore Its Dynamics. Journal of Fungi. 2022; 8(8):846. https://doi.org/10.3390/jof8080846
Chicago/Turabian StyleFernandes, José Alex Lourenço, Matheus da Silva Zatti, Thales Domingos Arantes, Maria Fernanda Bezerra de Souza, Mariana Marchi Santoni, Danuza Rossi, Cleslei Fernando Zanelli, Xiang-Qin Liu, Eduardo Bagagli, and Raquel Cordeiro Theodoro. 2022. "Cryptococcus neoformans Prp8 Intein: An In Vivo Target-Based Drug Screening System in Saccharomyces cerevisiae to Identify Protein Splicing Inhibitors and Explore Its Dynamics" Journal of Fungi 8, no. 8: 846. https://doi.org/10.3390/jof8080846