the cell surface and transport activity are reduced. The prolonged PKC/Nedd4‐2 activation results in enhanced OAT internalization from the cell surface to the intracellular endosomes and subsequent degradation in proteolytic systems. Overexpression of Nedd4‐2/C821A, a ligase‐dead mutant of Nedd4‐2, or the knockdown of endogenous Nedd4‐2 with Nedd4‐2‐specific siRNA evaded the PKC‐induced change in OAT ubiquitination, trafficking, and function in cultured cells [82, 91, 93]. On the other hand, serum and glucocorticoid‐regulated kinase 1 (Sgk1) phosphorylated Nedd4‐2 on Ser327 in cultured cells, which weakened the interaction between OATs and Nedd4‐2 and decreased OAT ubiquitination, leading to increased OAT transport activity [94]. Sgk2, an isoform of Sgk1, impaired the binding between OATs and Nedd4‐2 and decreased OAT ubiquitination, leading to elevated OAT cell surface expression and transport activity in cultured cells [95, 96]. In summary, PKC (negative regulation) and Sgk1/2 (positive regulation) exert opposite effects on OAT trafficking to the cell membrane and transport activity through phosphorylation of Nedd4‐2 at distinct sites. Nedd4‐2 serves as a central switch in these regulations.
Ubiquitination is a dynamic and reversible process. Deubiquitination is catalyzed by deubiquitinating enzymes (DUBs) and removes ubiquitin molecule(s) from the substrate protein [97, 98]. Deubiquitination and ubiquitination form a dynamic and opposing network, which is involved in a variety of physiological and pathological processes [99–102]; ~100 DUBs have been identified to date and are grouped into two classes: metalloproteases and cysteine proteases. Metalloproteases and cysteine proteases can be further subdivided into five subfamilies: ubiquitin‐specific proteases (USP), ubiquitin C‐terminal hydrolases (UCH), Jab1/Mpn/Mov34 (JAMM) enzymes, Machado‐Joseph domain proteases (MJD), and ovarian tumor proteases (OTU). Each of these subfamilies exhibits different structures and specificities toward distinct ubiquitin linkages [103–105]. USP8, a member of USP family, increased OAT cell surface expression and transporter activity in cultured cells, stemming from decreased OAT internalization, degradation, and ubiquitination. Knocking down the endogenous USP8 with USP8‐specific siRNA led to an increase in OAT ubiquitination, which correlated with reduced OAT transport activity [106]. In summary, ubiquitination and deubiquitination combine to help regulate OAT trafficking and transport activity (Fig. 4.2).
During the past few years, positive and negative crosstalk between different PTMs has been explored. Positive crosstalk is when one PTM serves as a signal for the modification of a second PTM, whereas negative crosstalk is when one PTM directly competes with another PTM or indirectly masks the recognition site for another PTM. The interplay between the PTMs that occur on the same type of amino acid residue(s) has attracted research attention because of its potential to regulate a wide array of cellular functions. One key example of negative crosstalk occurs between ubiquitination and SUMOylation, in which both ubiquitin and small ubiquitin‐like modifier (SUMO) are covalently attached to the lysine residue(s) of the target protein. Ubiquitin and SUMO can either conjugate to the same lysine residue(s) in the substrate protein in a competitive manner or conjugate to different lysine residues in the target protein. Under both circumstances, SUMOylation may preclude the ubiquitin‐mediated trafficking of the target protein [107]. It has been reported that the enhanced OAT SUMOylation induced by PKA activation occurs in parallel with a decrease in OAT ubiquitination, leading to an increased rate of OAT recycling and decreased rate of OAT degradation, without affecting the internalization rate of OATs. Therefore, SUMOylation and ubiquitination may coordinately regulate OAT trafficking and transport activity through negative crosstalk [108].
FIGURE 4.2 Post‐translational modifications of OATs. Ubiquitination and deubiquitination combine to regulate the expression of OATs at the cell surface, and thus their function. Ub: ubiquitin.
Several hormones and chemicals have been shown to regulate OAT trafficking through the protein kinases/Nedd4‐2 signaling pathway. Angiotensin II, an endogenous hormone, activated PKC/Nedd4‐2 pathway, which led to an increased rate of OAT internalization and therefore a reduction in OAT transport activity [109, 110]. AG490, a specific inhibitor of the Janus tyrosine kinase 2 (JAK2), reduced Nedd4‐2 phosphorylation at tyrosine residue(s), resulting in enhanced interaction between OAT and Nedd4‐2 and enhanced OAT ubiquitination, which led to a reduction in OAT cell surface expression and transport activity in cultured cells. Moreover, AG490 also increased the degradation rate of OATs. On the other hand, the inhibition effect of AG490 on OATs was diminished by knocking down the endogenous Nedd4‐2 using Nedd4‐2‐specific siRNA [111]. Dexamethasone, an upstream hormone of Sgk1, increased Nedd4‐2 phosphorylation, leading to stimulated OAT expression and transport activity in cultured cells [112]. Insulin, an endogenous hormone, phosphorylated Nedd4‐2 on Ser327 and impaired the interaction between OAT and Nedd4‐2, resulting in the upregulation of OAT expression and transport activity. Knocking down the endogenous Nedd4‐2 with Nedd4‐2‐specific siRNA diminished the upregulation on OATs induced by insulin [113].
4.4 OAT SUBSTRATES AND DRUG–DRUG (DRUG–METABOLITE) INTERACTION
4.4.1 Substrates
Multiple reviews describe the OAT substrates in great detail [23, 24, 114]. Here, we provide some general information. OATs handle a remarkably chemically diverse set of small molecule organic anions, including important and commonly prescribed pharmaceuticals (e.g., penicillins, cephalosporins, antiretrovirals, antihypertensives, antineoplastics, and diuretics), endogenous metabolites (e.g., cortisol and estrone‐3‐sulfate), and hormones, as well as environmental contaminants and toxicants (e.g., conjugates of organic mercury, perfluoroctanoic acid, ochratoxin A, and aristocholic acid), among others [24]. Because of the broad spectrum nature of the substrates, there are several substrates that are commonly used as tracers in published studies mostly performed in vitro assays. Some of the tracer/substrates include glutarate, PAH (para‐aminohippurate, a synthetic molecule), estrone sulfate, and more recently fluorescent compounds of fluorescein, 6‐carboxyfluorecein, and 5‐carboxyfluorecein.
4.4.2 Substrate Specificity
In spite of the apparent multi‐specific nature of the OATs, there exist specific structural/molecular determinants within their substrates, which help target them for elimination via a particular OAT. This view is supported by studies of knockouts of Oat1 and Oat3, which have revealed altered handling of a variety of pharmaceuticals and toxins (Table 4.2). For example, while in vitro studies suggested that PAH is handled by the majority of Oats, analysis of the Oat1 knockout animal found that in vivo handling of this “prototypical” Oat substrate was largely dependent on Oat1 [124, 125]. As expected, the handling of a number of drugs is affected in the Oat1 and Oat3 knockout animals or tissues (e.g., kidney, choroid plexus) derived from them. These include diuretics, antibiotics, antivirals, and methotrexate [38,115–117,125–128]. The knockout of Oat1 protects the kidney from injury mediated by mercury conjugates, suggesting that Oat1 is the primary renal transporter of this environmental toxin [119]. Certain metabolites such as urate appear to be handled in concert by Oats, Rst/Urat1, and other Slc and/or Abc transporters [38, 122]. In addition, metabolomics analysis of urine and plasma samples from Oat1‐null and wild‐type mice identified dozens of small molecule metabolites with altered concentration in the mutant mice [120, 123, 129]. However, the concentrations of these metabolites were not altered in Oat3‐null mice [125], providing support for the notion that Oat1 and Oat3 each have its own substrate spectrum.
TABLE 4.2 Characterization of Oat1/Oat3 knockout mice with respect to drugs, toxins, and endogenous metabolites
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