Dataset Information

Proteomics analysis of a new Cas9-targeting PROTAC molecule

ABSTRACT: Scientists have developed several methods to control the activity of Cas protein in a timely way and hence reduce off-target effects, including anti-CRISPR proteins and small molecule inhibitors. The PROTAC is a new conception of using the natural ubiquitin-proteasome system to degrade the protein of interest in drug design and development. Here, we engineered Cas proteins (Cas9, Cas12, and Cas13) by inserting a Phe-Cys-Pro-Phe (FCPF) amino acid sequence (also known as the π-clamp system) and demonstrate that the modified CasFCPF proteins could be labeled in live cells by perfluoroaromatics carrying FITC fluorophore or were degraded by perfluoroaromatics-functionalized PROTAC (PROTAC-FCPF). Proteome-wide analysis of PROTAC-FCPF-mediated Cas9FCPF protein degradation revealed high target specificity, suggesting a wide application of chemically induced proximity in combination with site-specific protein modification in the regulation of protein stability, activity and functionality.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Georg Tascher

LAB HEAD: Xinlai Cheng

PROVIDER: PXD022199 | Pride | 2022-10-18

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20200918_GT_XC_MUT_CTRL_1_B1.raw	Raw
	20200918_GT_XC_MUT_CTRL_1_B2.raw	Raw
	20200918_GT_XC_MUT_CTRL_1_B3.raw	Raw
	20200918_GT_XC_MUT_CTRL_1_B4.raw	Raw
	20200918_GT_XC_MUT_CTRL_1_B5.raw	Raw

Items per page:

1 - 5 of 58

Publications

A Chemical Toolbox for Labeling and Degrading Engineered Cas Proteins.

Gama-Brambila Rodrigo A RA Chen Jie J Dabiri Yasamin Y Tascher Georg G Němec Václav V Münch Christian C Song Guangqi G Knapp Stefan S Cheng Xinlai X

JACS Au 20210512 6

The discovery of clustered regularly interspaced short palindromic repeats and their associated proteins (Cas) has revolutionized the field of genome and epigenome editing. A number of new methods have been developed to precisely control the function and activity of Cas proteins, including fusion proteins and small-molecule modulators. Proteolysis-targeting chimeras (PROTACs) represent a new concept using the ubiquitin-proteasome system to degrade a protein of interest, highlighting the signific ...[more]

PMID: 34467332

Similar Datasets

Project description:CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism. 4 Samples: Protein-associated small RNAs

Project description:Aspergillus species are a leading cause of invasive fungal infections. As resistance to first-line therapy involving triazole antifungal agents is rising, second-line therapy with echinocandins is expanding. Resistance to echinocandins is well-established to result from amino acid substitutions in the echinocandin drug target β-(1,3)-D-glucan synthase encoded by the fks1 gene. Recently, we identified several high MIC clinical isolates of A. fumigatus from patients failing echinocandin therapy that did not contain any mutation in the fks1 gene, indicating that echinocandin resistance in these isolates results from an undefined mechanism. To explore possible new mechanisms of resistance, we used a lab-derived strain, RG101, with a nearly identical susceptibility phenotype, as a model system. This strain does not contain fks1 mutations but showed prominent resistance to the echinocandin class drug caspofungin (CAS), while remaining sensitive to other echinocandins, azoles and polyenes. Glucan synthase isolated from RG101 was fully sensitive to drug. Yet, exposure to CAS during its growth yielded a modified enzyme that was insensitive (4-log orders) in kinetic inhibition assays to CAS, as well as other echinocandins. This induction of cross-resistance by CAS was also observed in clinical isolates. To determine the nature of a presumptive posttranslational modification (PTM) of the enzyme, we analyzed whole enzyme PTMs, including the known hot-spot regions, for methylation, acetylation and phosphorylation. While we did not identify any PTMs linked to resistance, analysis of the lipid microenvironment of CAS-induced resistant enzyme revealed a prominent increase in the abundance of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to sensitive enzyme in in vitro kinetic inhibition assays recapitulated the CAS insensitivity of the cellular-derived enzyme. To further examine induction of drug-induced resistance, we used an in vitro assay to demonstrate that CAS, but not other echinocandin class drugs, prominently induced the production of mitochondrial-derived Reactive Oxygen Species (ROS) in A. fumigatus. RNASeq evaluation of whole cells confirmed a ROS signature in cells treated with CAS. Dampening the formation of ROS by antimycin A or thiourea eliminated the induction of drug resistance by CAS. We conclude that CAS-induced formation of ROS promotes a cellular stress response that alters the composition of plasma membrane lipids surrounding glucan synthase, changing its enzymatic properties to make it insensitive to echinocandins. This stress-induced response constitutes a novel mechanism of echinocandin resistance in Aspergillus, with implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.