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Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-?B.

ABSTRACT: The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor ?-light-chain-enhancer of activated B cells (NF-?B) transcription factor subunits, dampening activation of the NF-?B signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-?B subunits, including p65, RelB, and cRel but not NF-?B1 and NF-?B2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-?B subunits. Mutational analysis of NF-?B subunits revealed that a single nonconserved residue in NF-?B1 and NF-?B2 that corresponds to the P1' residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1' residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-?B subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-?B activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.

SUBMITTER: Jennings E

PROVIDER: S-EPMC6166728 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-κB.

Jennings Elliott E Esposito Diego D Rittinger Katrin K Thurston Teresa L M TLM

The Journal of biological chemistry 20180726 39

The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during <i>Salmonella</i> infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel ...[more]

PMID: 30049795

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Project description:Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by neurotropic polyomavirus, JC virus (JCV), a virus that causes lytic infection of CNS glial cells. After primary infection, JCV is controlled by the immune system but virus persists asymptomatically. Rarely, when immune function is impaired, it can reemerge to cause PML. The mechanisms of JCV persistence and reactivation are not well understood but our earlier work implicated epigenetic control by protein acetylation since histone deacetylase inhibitors such as trichostatin A (TSA) strongly stimulate JCV transcription. Since both TNF-α and TSA activate JCV transcription via the same unique NF-κB site in the JCV control region, we investigated a role for acetylation of NF-κB in JCV regulation. A site-directed mutagenesis strategy was employed targeting the known lysine acetylation sites of NF-κB p65: K218, K221, and K310. We individually mutated each lysine to arginine, which cannot be acetylated and retains a positive charge like lysine. K218R and K221R impaired transactivation of JCV early promoter transcription either alone or combined with TSA treatment or coexpression of acetyltransferase transcriptional coactivator p300 but K310R was largely without effect. Mutation of lysine to glutamine gives mutants with a negative charge like acetyllysine. However, K218Q and K221Q showed impaired activity and only K310Q showed enhanced transactivation. NF-κB acetylation can regulate several aspects of the process of activation including complex formation with IκB, translocation to the nucleus, and DNA binding and transcriptional activation. Cell fractionation studies revealed that the mutants had no defect in translocation to the nucleus whereas gel shift studies revealed reduced binding to the JCV NF-κB site. Thus, acetylation regulates NF-κB p65 activity toward JCV at the level of p65 binding to the JCV control region and activation of JCV transcription.

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Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-?B.

Publications

Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-κB.

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