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A method for investigating protein-protein interactions related to salmonella typhimurium pathogenesis.

ABSTRACT: We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). This method includes (i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques, (ii) in vivo cross-linking with formaldehyde, (iii) tandem affinity purification of bait proteins under fully denaturing conditions, and (iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions and permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of noncross-linked proteins to bait proteins. Two different negative controls were employed to eliminate the possibility of identifying background and nonspecific proteins as interacting partners, especially those caused by nonspecific binding to the stationary phase used for protein purification. In an initial demonstration of this approach, we tagged three Salmonella proteinsHimD, PduB and PhoPwith known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified for each bait protein, including the known binding partners such as HimA for HimD, as well as unexpected binding partners. Our results suggest that novel protein-protein interactions identified may be critical to pathogenesis by Salmonella.

SUBMITTER: Chowdhury SM

PROVIDER: S-EPMC2720628 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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A method for investigating protein-protein interactions related to salmonella typhimurium pathogenesis.

Chowdhury Saiful M SM Shi Liang L Yoon Hyunjin H Ansong Charles C Rommereim Leah M LM Norbeck Angela D AD Auberry Kenneth J KJ Moore Ronald J RJ Adkins Joshua N JN Heffron Fred F Smith Richard D RD

Journal of proteome research 20090301 3

We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). This method includes (i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques, (ii) in vivo cross-linking with formaldehyde, (iii) tandem affinity purification of bait proteins under fully denaturing conditions, and (iv) identification of the proteins cross-linked to the ba ...[more]

PMID: 19206470

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Project description:In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca2+.

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A method for investigating protein-protein interactions related to salmonella typhimurium pathogenesis.

Publications

A method for investigating protein-protein interactions related to salmonella typhimurium pathogenesis.

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