A Functional Protein Microarray Approach to Characterizing Posttranslational Modifications on Lysine Residues

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 -processes. 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.

Global identification of CobB interactors by an <italic>Escherichia coli</italic> proteome microarray

“…Proteome microarrays have been successfully applied in a variety of studies, such as biomarker discovery, cell surface marker/glycosylation profiling, post-translational modifications (PTMs), protein -protein, protein -lipid, protein -DNA, protein -small compound, and protein-peptide interactions, and enzyme substrates identification [16][17][18][19][20]. Recently, a proteome microarray carrying 4000 E. coli proteins has been constructed.…”

“…Protein microarrays are miniaturized, parallel assay systems that are constructed by spotting hundreds to thousands of affinity-purified proteins on a solid surface at high density [14][15][16][17]. Proteome microarrays have been successfully applied in a variety of studies, such as biomarker discovery, cell surface marker/glycosylation profiling, post-translational modifications (PTMs), protein -protein, protein -lipid, protein -DNA, protein -small compound, and protein-peptide interactions, and enzyme substrates identification [16][17][18][19][20].…”

Integrated Strategies to Gain a Systems-Level View of Dynamic Signaling Networks

“…Huang et al, 2004; Kung et al, 2009). Importantly, because they are also amenable to activity assays designed to determine enzyme-substrate relationships, functional protein microarrays have been particularly useful for determining the global substrate specificity of key signaling enzymes(Cox et al, 2015; Jeong, Rho, & Zhu, 2011; Lu, Lin, Boeke, & Zhu, 2013; Sun et al, 2016). For example, to gain a better understanding of the organization of human phosphorylation networks, we recently examined the global substrate profiles of 289 unique human kinases using functional protein microarrays composed of ~4,200 full-length human proteins (Newman et al, 2013).…”

Tools used to study how protein complexes are assembled in signaling cascades

“…Several adaptations of the protein array system, such as suspension bead arrays, have the potential to circumnavigate this problem 36 and studies investigating post-translational modifications of particular residues such as lysine have been successful and are being used to study ubiqutin (Ub) ligases, SUMOylation E3 ligases and acetyltransferases. 37 Advances in peptide synthesis on nitrocellulose support have also helped overcome some of these challenges and is discussed later in the review. Many laboratories still rely on more classical approaches such as synthetic lethality, 38 Electrophoretic Mobility Shift Assays (EMSAs) and Far-protein gel blotting.…”