Dataset Information

Identification of YqfB enzyme expressed in colorectal cancer cell line HCT116 using LC-MS/MS proteomics

ABSTRACT: Gene-directed enzyme prodrug therapy is an emerging strategy for cancer treatment based on the delivery of a gene that encodes an enzyme that is able to convert a prodrug into a potent cytotoxin exclusively in target cancer cells. The aim of this study is to determine the capability of bacterial amidohydrolases to activate novel prodrugs and the effect such system has on the viability of eukaryotic cancer cells. HCT116 cancer cell line was subjected to LC-MS/MS proteomic analysis to confirm bacterial YqfB amidohydrolase expression at protein level.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Gediminas Plakys

LAB HEAD: Rolandas Meškys

PROVIDER: PXD045918 | Pride | 2024-01-26

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	HCT116_YqfB_Ni-NTA_10ul.raw.zip	Raw
	HCT116_YqfB_Ni-NTA_10ul.zip	Other
	HCT116_YqfB_Ni-NTA_10ul_2.raw.zip	Raw
	HCT116_YqfB_Ni-NTA_10ul_2.zip	Other
	checksum.txt	Txt

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Publications

Bacterial amidohydrolases and modified 5-fluorocytidine compounds: Novel enzyme-prodrug pairs.

Preitakaitė Viktorija V Barasa Povilas P Aučynaitė Agota A Plakys Gediminas G Koplūnaitė Martyna M Zubavičiūtė Simona S Meškys Rolandas R

PloS one 20231130 11

Gene-directed enzyme prodrug therapy is an emerging strategy for cancer treatment based on the delivery of a gene that encodes an enzyme that is able to convert a prodrug into a potent cytotoxin exclusively in target cancer cells. However, it is limited by the lack of suitable enzyme variants and a scarce choice of chemical bonds that could be activated. Therefore, this study is aimed to determine the capability of bacterial amidohydrolases YqfB and D8_RL to activate novel prodrugs and the effec ...[more]

PMID: 38032917

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Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.