Elsevier

Pharmacology & Therapeutics

Volume 192, December 2018, Pages 88-99
Pharmacology & Therapeutics

Post-translational modifications of transporters

https://doi.org/10.1016/j.pharmthera.2018.06.013Get rights and content

Abstract

Drug transporter proteins are critical to the distribution of a wide range of endogenous compounds and xenobiotics such as hormones, bile acids, peptides, lipids, sugars, and drugs. There are two classes of drug transportersā€“ the solute carrier (SLC) transporters and ATP-binding cassette (ABC) transporters ā€“which predominantly differ in the energy source utilized to transport substrates across a membrane barrier. Despite their hydrophobic nature and residence in the membrane bilayer, drug transporters have dynamic structures and adopt many conformations during the translocation process. Whereas there is significant literature evidence for the substrate specificity and structure-function relationship for clinically relevant drug transporters proteins, there is less of an understanding in the regulatory mechanisms that contribute to the functional expression of these proteins. Post-translational modifications have been shown to modulate drug transporter functional expression via a wide range of molecular mechanisms. These modifications commonly occur through the addition of a functional group (e.g. phosphorylation), a small protein (e.g. ubiquitination), sugar chains (e.g. glycosylation), or lipids (e.g. palmitoylation) on solvent accessible amino acid residues. These covalent additions often occur as a result of a signaling cascade and may be reversible depending on the type of modification and the intended fate of the signaling event. Here, we review the significant role in which post-translational modifications contribute to the dynamic regulation and functional consequences of SLC and ABC drug transporters and highlight recent progress in understanding their roles in transporter structure, function, and regulation.

Introduction

Transporter proteins are integral membrane proteins critical to the uptake, distribution, and excretion of endogenous compounds and xenobiotics such as nutrients, hormones, bile acids, peptides, lipids, sugars, and drugs (Cesar-Razquin et al., 2015). They can be broadly categorized into two superfamilies: the SoLute Carrier (SLC) transporters comprising over 400 integral membrane proteins subdivided into 50+ families and the ATP-Binding Cassette (ABC) superfamily, consisting of 7 families (ABCA through ABCG). Membrane transporters are inherently hydrophobic and large transmembrane domains span the cellular membrane (Harris & Booth, 2012; Harris, Findlay, Simms, Liu, & Booth, 2014). Despite their hydrophobicity, transporters have dynamic structures, and can adopt conformations that are not readily captured via X-ray crystallography. Therefore, specific proteoforms identified biochemically and computationally are often relied upon for mechanistic insight into the function of these proteins (Schlessinger, Khuri, Giacomini, & Sali, 2013; Schlessinger, Yee, Sali, & Giacomini, 2013; Shaikh et al., 2013). Post-translational modifications (PTMs) are regulators of these structural events and are critical for the transporters' structure, function, and regulation within the confines of the lipid environment. The hydrophilic loops and termini that face the intracellular milieu may be accessible for these modifications if the exposed amino acid side chains are solvent accessible, providing a mechanism in which the chemical nature of an amino acid can be altered (Walsh, Garneau-Tsodikova, & Gatto, 2005).

While there are 400+ types of PTMs that have been identified to date, the most common variants that are known to play a role (or, those that have been actively investigated) in the regulation of transporters include phosphorylation, glycosylation, and ubiquitination. Biochemically, these modifications diversify the nature of the amino acid peptide-backbone or side chain through the addition of small chemical groups (e.g. phosphates), lipids (e.g. palmitic acid), carbohydrates (e.g. mannose), small proteins (e.g. SUMO), among other entities (Duan & Walther, 2015; Hunter, 2007; Korkuc & Walther, 2017; Prabakaran, Lippens, Steen, & Gunawardena, 2012; Schlessinger, Khuri, et al., 2013; Schlessinger, Yee, et al., 2013; Shaikh et al., 2013; Walsh et al., 2005). Most, if not all, eukaryotic proteins undergo PTMs, and the likelihood for a given modification to occur is driven by the amino acid sequence, the structural and chemical constraints of the protein surface, and the availability of the necessary protein machinery and precursors to facilitate the modification (Fig. 1) (Duan & Walther, 2015; Korkuc & Walther, 2017). For transporters, this likelihood is further complicated by lipid-protein interactions driven by the buried transmembrane domains, which are obligatory to the functional expression of the protein. As such, solvent accessible residues located within the hydrophilic loops and termini of the membrane transporter often contain canonical consensus motifs (Table 1) that serve as recognition sites for not just the PTM, but also for the required adaptor proteins and enzymes needed to facilitate the modification (Amanchy et al., 2007; Dietrich & Ungermann, 2004; Guan & Fierke, 2011; Knorre, Kudryashova, & Godovikova, 2009; Rodriguez, Dargemont, & Hay, 2001; Walsh et al., 2005). While common consensus motifs are conserved across the proteome, there also are non-canonical recognition motifs that have been identified under physiologically relevant contexts (Kaneko, Joshi, Feller, & Li, 2012).

PTMs have been shown to influence transporter kinetics, both directly and indirectly (Xu & You, 2017). They do not just regulate the innate structure-function relationship driven by a transporter's global architecture, but rather are also able to regulate this relationship down to the resolution of the structural events occurring at a residue level. From this level, PTMs are able to modulate a transporter's function, expression, efficiency, structure, fate, interactions, and more. Additionally, the complexity of the response of PTM modulation is a function of the extent of modifications a transporter is able to accept and the availability of the environmental cues to signal for the post-translational event (Table 2) (Hunter, 2007; Kaneko et al., 2012; Walsh et al., 2005). This results in an exponential increase in specific ā€œspeciesā€ of a given transporter, referred to as proteoforms (Toby, Fornelli, & Kelleher, 2016), which can provide deeper mechanistic understanding in transporter biology.

The consequence of PTM modulation has been investigated for a wide range of SLC and ABC transporters, including but not limited to those described in the sections below. The purpose of this review is to provide a broad overview of the roles of PTMs in regulating transporters in higher vertebrates and humans, and consequently is not intended to be a comprehensive list of all of the PTMs identified to-date. Consequently, a diverse set of examples was selected from the literature to support the overarching themes in the post-translational regulation of ABC and SLC transporters (Table 3).

Section snippets

Background

Protein phosphorylation is a reversible PTM that affects an estimated one third of the total human proteome. Protein phosphorylation involves the addition of a terminal phosphate group to a free hydroxyl on the side chains of primarily serine and threonine residues, and to a lesser extent on tyrosine residues. The addition of the phosphate group is catalyzed by kinases, which are specific to the residue being modified (e.g. tyrosine kinase) and to the specific signal cascade initiated (e.g. the

Background

More than half of the mammalian proteome and nearly all SLC transporters are glycosylated, with N-glycosylation predominating. N-glycosylation is the process of enzymatically adding an oligosaccharide to extracellular asparagine residues and is often signaled by the motif N-X-S/T, where X is any amino acid except proline (P) or aspartic acid (D). N-glycosylation is a highly variable PTM, which is impacted by the availability of the sugar monomers at the time of protein folding and processing.

Background

Lysine acetylation (Table 2) is a reversible modification that occurs on the Īµ-amino group of intracellular lysines. It has been implicated as a dynamic post-translational response to changes in metabolism, and modified sites may be conserved between species. Lundby and colleagues (Lundby et al., 2012) recently carried out an extensive proteomic analysis to define tissue and cellular acetylation patterns. Intriguingly, they found over 4000 unique acetylated proteins in 16 different rat organs.

Background

Palmitoylation involves the enzymatic addition of the C16 saturated fatty acid, palmitic acid, to a free cysteine that lies adjacent to the protein-lipid interface (Dietrich & Ungermann, 2004; Guan & Fierke, 2011). Palmitoylation differs greatly from the relatively static nature of similar lipid modifications, such as myristoylation, in that it is highly dynamic. It has been observed that the reversal of protein palmitoylation can occur at variable rates from minutes to days depending on the

Background

The ubiquitin-proteosome pathway is a physiological system for the signaling and subsequent degradation of unstable proteins via the proteasome. Ubiquitin (Ub), a small, 8ā€ÆkDa polypeptide, is covalently linked to accessible lysine residues via a three-step process. Initiation of the reaction is carried out by a ubiquitin-activating enzyme (E1). Following activation, Ub structurally associates with a carrier protein (E2) which, coupled with a ubiquitin-protein-ligase (E3), modifies the target

SUMOylation as a gatekeeper of SLC transporter intracellular pools

The expression of the EAAT2 (SLC1A2) is not just influenced by ubiquitination, but also is influenced by the closely related PTM, SUMOylation (Foran et al., 2011; Foran, Rosenblum, Bogush, Pasinelli, & Trotti, 2014; Gibb et al., 2007; Sorkina et al., 2006). Like ubiquitin, SUMO (Small Ubiquitin-like MOdifier) is a reversible modification that involves the enzymatic addition of a small protein SUMO to an accessible lysine (Sampson, Wang, & Matunis, 2001). EAAT2 is modified at K580 under normal

Conclusions

While the modulation of signaling mechanisms is readily recognized as a critical regulatory facet for the functional integrity of homeostatic processes, there is still limited information on how they influence transporter proteins. ABC and SLC transporters are vital to the efficient tissue handling of endogenous and exogenous solutes. The two families combined represent over 500 functional proteins that are integral in almost every single homeostatic process in the human body. The two families

Conflict of interest statement

LCC and PWS declare no conflicts of interest. KMH is an employee of Eli Lilly & Company.

Acknowledgments

This work was funded in part by a grant from the National Institutes of Health, NIDDK award #DK61425 (to PWS).

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