Peter Jorth, Benjamin J. Staudinger, Xia Wu, Katherine B. Hisert, Hillary Hayden, Jayanthi Garudathri, Christopher L. Harding, Matthew C. Radey, Amir Rezayat, Gilbert Bautista, William R. Berrington, Amanda F. Goddard, Chunxiang Zheng, Angus Angermeyer, Mitchell J. Brittnacher, Jacob Kitzman, Jay Shendure, Corinne L. Fligner, John Mittler, Moira L. Aitken, Colin Manoil, James E. Bruce, Timothy L. Yahr, Pradeep K. Singh.
Chemoresistance is a common mode of therapy failure for many cancers. Tumours develop resistance to chemotherapeutics through a variety of mechanisms, with proteins serving pivotal roles. Changes in protein conformations and interactions affect the cellular response to environmental conditions contributing to the development of new phenotypes. The ability to understand how protein interaction networks adapt to yield new function or alter phenotype is limited by the inability to determine structural and protein interaction changes on a proteomic scale. Here, chemical crosslinking and mass spectrometry were employed to quantify changes in protein structures and interactions in multidrug-resistant human carcinoma cells. Quantitative analysis of the largest crosslinking-derived, protein interaction network comprising 1,391 crosslinked peptides allows for ‘edgotype’ analysis in a cell model of chemoresistance. We detect consistent changes to protein interactions and structures, including those involving cytokeratins, topoisomerase-2-alpha, and post-translationally modified histones, which correlate with a chemoresistant phenotype.
In pathogenic Gram-negative bacteria, interactions among membrane proteins are key mediators of host cell attachment, invasion, pathogenesis, and antibiotic resistance. Membrane protein interactions are highly dependent upon local properties and environment, warranting direct measurements on native protein complex structures as they exist in cells. Here we apply in vivo chemical cross-linking mass spectrometry, to reveal the first large-scale protein interaction network in Pseudomonas aeruginosa, an opportunistic human pathogen, by covalently linking interacting protein partners, thereby fixing protein complexes in vivo. A total of 626 cross-linked peptide pairs, including previously unknown interactions of many membrane proteins, are reported. These pairs not only define the existence of these interactions in cells but also provide linkage constraints for complex structure predictions. Structures of three membrane proteins, namely, SecD-SecF, OprF, and OprI are predicted using in vivo cross-linked sites. These findings improve understanding of membrane protein interactions and structures in cells.
Genetically-susceptible bacteria become antibiotic tolerant during chronic infections, and the mechanisms responsible are poorly understood. One factor that may contribute to differential sensitivity in vitro and in vivo is differences in the time-dependent tobramycin concentration profile experienced by the bacteria. Here, we examine the proteome response induced by sub-inhibitory concentrations of tobramycin in P. aeruginosa cells grown under planktonic conditions. These efforts revealed increased levels of heat shock proteins and proteases were present at higher dosage treatments (0.5 and 1 micrograms/ml), while less dramatic at 0.1 micrograms/ml dosage. In contrast, many metabolic enzymes were significantly induced by lower dosages (0.1 and 0.5 micrograms/ml), but not at 1 micrograms/ml dosage. Time course proteome analysis further revealed that the increase of heat shock proteins and proteases was most rapid from 15 min to 60 min, and the increased levels sustained till 6 hours (last time point tested). Heat shock protein IbpA exhibited the greatest induction by tobramycin, up to 90-fold. Nevertheless, deletion of ibpA did not enhance sensitivity to tobramycin. It seemed possible that the absence of sensitization could be due to redundant functioning of IbpA with other proteins that protect cells from tobramycin. Indeed, inactivation of two heat shock chaperones/proteases in addition to ibpA in double mutants (ibpA/clpB, ibpA/PA0779 and ibpA/hslV) did increase tobramycin sensitivity. Collective, these results demonstrate the time- and concentration-dependent nature of the P. aeruginosa proteome response to tobramycin, and that proteome modulation and protein redundancy are protective mechanisms to help bacteria resist antibiotic treatments.