Abstract
Functional protein microarrays offer a versatile platform to address diverse biological questions. Printing individually purified proteins in a spatially addressable format makes it straightforward to investigating binary interactions. To connect substrates to their upstream modifying enzymes, such as kinases, ubiqutin (Ub) ligases, SUMOylation E3 ligases, and acetyltransferases, is an especially daunting task using traditional methodologies. In recent years, regulation via various types of posttranslational modifications (PTMs) on lysine residues is emerging as an important mechanism(s) underlining diverse biological Âprocesses. Our group has been developing and applying functional protein microarrays constructed for different model organisms to globally identify enzyme–substrate interactions with a focus on lysine PTMs. In particular, we have characterized the pleiotropic functions of a ubiquitin E3 ligase, Rsp5, via identification of its downstream substrates using a yeast proteome chip. Also, we have identified nonhistone substrates of the acetyltransferase NuA4 complex in yeast, and revealed that reversible acetylation on a metabolic enzyme affects a glucose metabolism and contributes to life span. In this chapter, we will provide detailed protocols for the investigation of ubiquitylation and acetylation. These protocols are generally applicable for different organisms.
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Acknowledgments
We thank Dr. Wendy Yap for critical comments and editing. This work is in part supported by the NIH.
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Jeong, J.S., Rho, HS., Zhu, H. (2011). A Functional Protein Microarray Approach to Characterizing Posttranslational Modifications on Lysine Residues. In: Wu, C. (eds) Protein Microarray for Disease Analysis. Methods in Molecular Biology, vol 723. Humana Press. https://doi.org/10.1007/978-1-61779-043-0_14
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DOI: https://doi.org/10.1007/978-1-61779-043-0_14
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