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Enzymatic processing of platinated RNAs.

ABSTRACT: The broadly prescribed antitumor drug cisplatin coordinates to DNA, altering the activity of cellular proteins whose functions rely upon sensing DNA structure. Cisplatin is also known to coordinate to RNA, but the effects of RNA-Pt adducts on the large number of proteins that process the transcriptome are currently unknown. In an effort to address how platination of an RNA alters the function of RNA processing enzymes, we have determined the influence of [Pt(NH(3))(2)](2+)-RNA adducts on the activities of 3'-->5' and 5'-->3' phosphodiesterases, a purine-specific endoribonuclease, and a reverse transcriptase. Single Pt(II) adducts on RNA oligonucleotides of the form (5'-U(6)-XY-U(5)-3': XY = GG, GA, AG, GU) are found to block exonucleolytic digestion. Similar disruption of endonucleolytic cleavage is observed, except for the platinated XY = GA RNA where RNase U2 uniquely tolerates platinum modification. Platinum adducts formed with a more complex RNA prevent reverse transcription, providing evidence that platination is capable of interfering with RNA's role in relaying sequence information. The observed disruptions in enzymatic activity point to the possibility that cellular RNA processing may be similarly affected, which could contribute to the cell-wide effects of platinum antitumor drugs. Additionally, we show that thiourea reverses cisplatin-RNA adducts, providing a chemical tool for use in future studies regarding cisplatin targeting of cellular RNAs.

SUBMITTER: Chapman EG

PROVIDER: S-EPMC3230263 | biostudies-other | 2010 Feb

REPOSITORIES: biostudies-other

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Enzymatic processing of platinated RNAs.

Chapman Erich G EG DeRose Victoria J VJ

Journal of the American Chemical Society 20100201 6

The broadly prescribed antitumor drug cisplatin coordinates to DNA, altering the activity of cellular proteins whose functions rely upon sensing DNA structure. Cisplatin is also known to coordinate to RNA, but the effects of RNA-Pt adducts on the large number of proteins that process the transcriptome are currently unknown. In an effort to address how platination of an RNA alters the function of RNA processing enzymes, we have determined the influence of [Pt(NH(3))(2)](2+)-RNA adducts on the act ...[more]

PMID: 20099814

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Project description:The intraglomerular renin-angiotensin system (RAS) is linked to the pathogenesis of progressive glomerular diseases. Glomerular podocytes and mesangial cells play distinct roles in the metabolism of angiotensin (ANG) peptides. However, our understanding of the RAS enzymatic capacity of glomerular endothelial cells (GEnCs) remains incomplete. We explored the mechanisms of endogenous cleavage of ANG substrates in cultured human GEnCs (hGEnCs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and isotope-labeled peptide quantification. Overall, hGEnCs metabolized ANG II at a significantly slower rate compared with podocytes, whereas the ANG I processing rate was comparable between glomerular cell types. ANG II was the most abundant fragment of ANG I, with lesser amount of ANG-(1-7) detected. Formation of ANG II from ANG I was largely abolished by an ANG-converting enzyme (ACE) inhibitor, whereas ANG-(1-7) formation was decreased by a prolylendopeptidase (PEP) inhibitor, but not by a neprilysin inhibitor. Cleavage of ANG II resulted in partial conversion to ANG-(1-7), a process that was attenuated by an ACE2 inhibitor, as well as by an inhibitor of PEP and prolylcarboxypeptidase. Further fragmentation of ANG-(1-7) to ANG-(1-5) was mediated by ACE. In addition, evidence of aminopeptidase N activity (APN) was demonstrated by detecting amelioration of conversion of ANG III to ANG IV by an APN inhibitor. While we failed to find expression or activity of aminopeptidase A, a modest activity attributable to aspartyl aminopeptidase was detected. Messenger RNA and gene expression of the implicated enzymes were confirmed. These results indicate that hGEnCs possess prominent ACE activity, but modest ANG II-metabolizing activity compared with that of podocytes. PEP, ACE2, prolylcarboxypeptidase, APN, and aspartyl aminopeptidase are also enzymes contained in hGEnCs that participate in membrane-bound ANG peptide cleavage. Injury to specific cell types within the glomeruli may alter the intrarenal RAS balance.

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Enzymatic processing of platinated RNAs.

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Enzymatic processing of platinated RNAs.

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