Protein interactions with metallothionein-3 promote vectorial active transport in human proximal tubular cells

Abstract

Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, β-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments

“…Actin and MT-3 have been linked in a number of studies [24][25][26][27][28]. These links motivated us to investigate the molecular properties underlying the actin-MT-3 interaction and to probe the role of metal ions in this interaction.…”

“…In addition to a role for MT-3 in astrocyte actin remodeling, MT-3 is implicated in cytoskeletal regulation during the doming of kidney proximal tubule cells [26,66]. The doming process in cultured cells is a model for vectorial active transport.…”

“…Motivated by the potential importance of protein-protein interactions in MT-3 function, several screens have been carried out to characterize MT-3's interactome [24][25][26]. Using immunoaffinity chromatography and tandem mass spectrometry, at least 12 proteins have been identified that bind to MT-3 [24,25].…”

“…Using immunoaffinity chromatography and tandem mass spectrometry, at least 12 proteins have been identified that bind to MT-3 [24,25]. Of particular interest to this work, the cytoskeletal protein actin (𝛽 and 𝛾 isoforms) was one of the proteins most often identified in complex with MT-3 in these screening studies [24][25][26]. Based on the list of MT-3-binding proteins, Armitage and coworkers proposed that MT-3 is secreted from astrocytes through its interaction with Rab3A, Exo84p, and 14-3-3 zeta and then transported into neurons through interaction with plasminogen (PGn) and enolase, where MT-3 could inhibit growth through its effect on the cytoskeleton and/or other interactions [25].…”

“…In addition to the appearance of actin in interaction screens, MT-3 and actin have been functionally linked in mouse astrocytes [27,28] and in human kidney proximal tubule cells [26]. In astrocytes, changes in the actin cytoskeleton that occur downstream of epidermal growth factor (EGF) treatment are abnormal in the absence of MT-3 [27].…”