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Differential alkylation of PHLPP2

ABSTRACT: To determine the accessibility of cysteines in purified PHLPP2, we assessed their reactivity with iodoacetamide under native conditions. After denaturing with urea and reducing potential disulfide bridges with TCEP, the remaining free cysteines were modified with methyl methanethiosulfonate. This 2-step differential alkylation experiment was compared to the 1-step alkylation reactions under fully denaturing conditions with iodoacetamide and methyl methanethiosulfonate.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Markus Hartl

LAB HEAD: Dr. Thomas A. Leonard

PROVIDER: PXD052551 | Pride | 2025-03-13

REPOSITORIES: Pride

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			Action	DRS
	20231130_E2_NEO5_Husremovic_Leonard_MPL_MUW_DF_95p.raw	Raw
	20231130_E2_NEO5_Husremovic_Leonard_MPL_MUW_IAM_1p.raw	Raw
	20231130_E2_NEO5_Husremovic_Leonard_MPL_MUW_MMTS_1p.raw	Raw
	FragPipe_search_result.zip	Other
	Raw_files_experiments_list.xlsx	Xlsx

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Project description:Alkylation damage to DNA occurs when cells encounter alkylating agents in the environment or when active alkylators are generated by nitrosation of amino acids in metabolic pathways. To cope with DNA alkylation damage, cells have evolved genes that encode proteins with alkylation-specific DNA repair activities. It is notable that these repair systems are conserved from bacteria to humans. In Escherichia coli, cells exposed to a low concentration of an alkylating agent, such as N-methyl-Nâ-nitro-N-nitrosoguanidine (MNNG) or methyl methanesulfonate (MMS), show a remarkable increase in resistance to both the lethal and mutagenic effects of subsequent high-level challenge treatments with the same or other alkylating agents. This increased resistance has been known as âadaptive responseâ to alkylation damage in DNA. To date, four genes have been identified as components of this response, ada, alkA, alkB and aidB. The ada gene encodes the Ada protein, which has the dual function of a transcriptional regulator for the genes involved in the adaptive response, and a methyltransferase that demethylates two methylated bases (O6meG and O4meT) and methylphosphotriesters produced by methylating agents in the sugar phosphate backbone. The differences between the wild-type and mutant strains were characterized at transcriptome levels. In addition, the global changes in gene expressions in response to alkylating agents (MMS), in E. coli K-12 W3110 and ada mutant strains were also analyzed. The analysis of time- and strain-dependent adaptive responses revealed the regulatory and physiological characteristics of the Ada-dependent adaptive response in E. coli. In order to examine the intracellular changes that are induced by the ada gene deletion in the MMS-untreated, normal growth condition, the expression levels of genes of ada mutant cells were compared with those of wild-type cells at the mid-log growth phase (at 0.5 h sampling point). Cells were cultivated at 37oC and 250 rpm in 100 mL of Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) in 250-mL Erlenmeyer flasks. Transcriptome analysis were also performed for the samples (E. coli wildtype and ada mutant strains) taken at 0.5, 1.5 and 3.9 h following MMS treatment for both MMS-treated and -untreated control cultures, and the expression levels were compared.

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Differential alkylation of PHLPP2

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