Details:

Alternative Title:
Free Radic Biol Med
Personal Author:
Gil, Magdalena ; Graña, Martín ; Schopfer, Francisco J. ; Wagner, Tristan ; Denicola, Ana ; ... More +
Gil, Magdalena ; Graña, Martín ; Schopfer, Francisco J. ; Wagner, Tristan ; Denicola, Ana ; Freeman, Bruce A. ; Alzari, Pedro M. ; Batthyány, Carlos ; Durán, Rosario ; Less -
Description:
PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.
Subject:
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Alkenes
Article
Bacterial Proteins
Catalytic Domain
Circular Dichroism
Fatty Acids
Mutagenesis, Site-Directed
Mycobacterium Tuberculosis
Nitrated Fatty Acids
Nitroalkene
Nitro Compounds
PknG
Protein-Serine-Threonine Kinases
Rubredoxin
Rubredoxins
Ser/Thr Kinase
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Source:
Free Radic Biol Med. 2013 ; 65:150-161.;
Pubmed ID:
23792274
Pubmed Central ID:
PMC4061738
Document Type:
Journal Article ;
Funding:
NIHR01-HL058115/HL/NHLBI NIH HHS/United States ; R01AT-006822/AT/NCCIH NIH HHS/United States ; P01 HL103455/HL/NHLBI NIH HHS/United States ; R01 HL058115/HL/NHLBI NIH HHS/United States ; R01 AT006822/AT/NCCIH NIH HHS/United States ; ... More +
NIHR01-HL058115/HL/NHLBI NIH HHS/United States ; R01AT-006822/AT/NCCIH NIH HHS/United States ; P01 HL103455/HL/NHLBI NIH HHS/United States ; R01 HL058115/HL/NHLBI NIH HHS/United States ; R01 AT006822/AT/NCCIH NIH HHS/United States ; R01 HL064937/HL/NHLBI NIH HHS/United States ; R01-HL64937/HL/NHLBI NIH HHS/United States ; P01-HL103455/HL/NHLBI NIH HHS/United States ; U17 CE002009/CE/NCIPC CDC HHS/United States ; Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:828d277c3625cf6fc9471eed9733aff4de4e42d9aab60a84958da91242255658
File Type:

Supporting Files

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