Getting down to details: disease-associated PTMs and proteoforms

27 Apr 2018 1:08 PM | Anonymous member (Administrator)

Michelle Hill, QIMR Berghofer Medical Research Institute, and The University of Queensland, Australia

Post-translational modifications (PTMs) on proteins can play a critical role in function by regulating protein structure, function and/or localization. Although consensus sites for some of the more well-studied PTMs have been established, PTMs are not predictable from the genetic code. Furthermore, PTMs can be either stably attached through the life of a protein, or reversibly added depending on stimulation. Hence, direct interrogation at the protein level is required to fully characterize PTM function in health and disease.

Several B/D-HPP teams have been investigating the biological roles of PTMs and their disease associations, with two recent publications in leading interdisciplinary journals.

Neil Kelleher and his team at Northwestern University used top-down proteomics to characterize proteoforms of KRAS, a gene frequently mutated in human cancer. The novel mass spectrometry-based whole protein assay enabled the team to investigate the effect of genetic mutations in KRAS on PTMs of the protein that are important for proper KRAS regulation. In collaboration with National Cancer Institute, the study, recently published in Proceedings of the National Academy of Science USA, quantified the percentage of mutant KRAS4b present in colorectal cancer tissue, and identified differences in the level of C-terminal carboxymethylation, which is critical for KRAS function. Such detailed characterization and quantification of KRAS proteoforms was only possible with the novel top down mass spectrometry-based assay. In the future, targeted assays such as this will be extended to characterize proteoforms within specific cell types to increase their sensitivity and selectivity and inform cancer prognosis and personalized therapy selection.

A collaboration between Jenny Van Eyk’s team at Cedars-Sinai Medical Center and Shengbing Wang and David Kass at Johns Hopkins University School of Medicine, determined the functional consequences of a redox-regulated PTM on glycogen synthase kinase 3b (GSK3b). S-nitrosylation is the attachment of nitric oxide to sulfhydryl residues of proteins, and is involved in redox-based cell signaling. The study, published in Circulation Research, reported the novel finding that GSK3b can be modified by S-nitrosylation at specific sites in models of heart failure and sudden cardiac death. S-nitrosylation of GSK3b overrides the classical phospho-regulation and sends it to the nucleus, away from its usual cytoplasmic location. The result is that the S-nitrosylated GSK3b now phosphorylates a completely different repertoire of proteins in the nucleus compared to its known cytoplasmic substrates, implying that drugs meant to target cytoplasmic GSK3b may inhibit complete different nuclear pathways if the GSK3b is S-nitrosylated. “Knowing the PTM status of drug targets and the functional effect is key to next generation therapies.” Says Jenny.

Together, these studies exemplify the work of B/D-HPP teams and clinical collaborators on apply and developing advanced proteomics methods to understanding disease. Future work of the B/D-HPP teams will be to translate the findings into the pathology or clinical chemistry laboratories, to make an impact on patients lives.

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