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Coupled Mutations-Enabled Glycerol Transportation in an Aquaporin Z Mutant.

ABSTRACT: Aquaporins are transmembrane channel proteins with key function being transportation of water or other small substrates. Escherichia coli Aqp Z transports water molecules only, whereas Glp F is permeable to glycerol. It is intriguing to explore the possibility to induce glycerol permeability in Aqp Z by targeted mutations. The Aqp Z mutants with mutated selectivity filter (SF) residues exhibit poor permeability for both glycerol and water. For addressing the complexity of protein systems, pair correlation information in protein sequence analyses is instructive to identify residues that are coupled by coevolution and motion. In this study, we analyze the correlation between residues and unravel the clustering patterns of coupled residues, beyond SF residues, in aquaglyceroporins (AQGPs). The identified coupled motifs are proposed to be sequenced into aquaporin (Aqp Z) to introduce glycerol permeability. These residues are located in the vicinity of SF region, C-loop, and M6-M7 linkage domain. Significant enlargement of SF pore size of the proposed Aqp Z mutant is observed by an all-atom replica exchange molecular dynamics simulation, which is critical to facilitate considerable glycerol passage as characterized in calculated free-energy landscapes. Clearly, the hidden connections among residues play crucial roles in water/glycerol selectivity. In contrast, single-site mutation-based scheme may even lead to undesirable effects in AQGPs, such as the blocking of water transportation by aromatic ?-stacked gate. As demonstrated in this work, the pair correlation analysis guided rational mutagenesis provides a feasible strategy to modulate proteins' functions.

SUBMITTER: Ping Z

PROVIDER: S-EPMC6641515 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Coupled Mutations-Enabled Glycerol Transportation in an Aquaporin Z Mutant.

Ping Zhi Z Zhou Feng F Lin Xin X Su Haibin H

ACS omega 20180412 4

Aquaporins are transmembrane channel proteins with key function being transportation of water or other small substrates. <i>Escherichia coli</i> Aqp Z transports water molecules only, whereas Glp F is permeable to glycerol. It is intriguing to explore the possibility to induce glycerol permeability in Aqp Z by targeted mutations. The Aqp Z mutants with mutated selectivity filter (SF) residues exhibit poor permeability for both glycerol and water. For addressing the complexity of protein systems, ...[more]

PMID: 31458647

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Project description:BackgroundDiuretic agents are widely used on the treatment of water retention related diseases, among which acetazolamide (AZA) acts originally as a carbonic anhydrase (CA) inhibitor. Aquaporin-1 (AQP1) being located in renal proximal tubules is required for urine concentration. Previously our lab has reported AZA putatively modulated AQP1. Aim of this study is to testify our hypothesis that regulating AQP1 may mediate diuretic effect of AZA.Methodology/principal findingsFor in vivo study, we utilized Sprague Dawley rats, as well as AQP1 knock-out (AQP1(-/-)) mice to examine urine volume, and human kidney-2 (HK-2) cell line was used for in vitro mechanism study. In our present study we found that AZA decreased CAs activity initially but the activity gradually recovered. Contrarily, diuretic effect was consistently significant. AQP1 protein expression was significantly decreased on day 7 and 14. By utilizing AQP1(-/-) mice, we found diuretic effect of AZA was cancelled on day 14, while urine volume continuously increased in wild-type mice. Surface plasmon resonance (SPR) results indicated AQP1 was physiologically bound by myosin heavy chain (MHC), immunoprecipitation and immunofluorescence results confirmed this protein interaction. In vitro study results proved AZA facilitated AQP1 translocation onto cell membrane by promoting interaction with MHC, dependent on ERK/ myosin light chain kinase (MLCK) pathway activation. MHC inhibitor BDM and ERK inhibitor U0126 both abolished above effect of AZA. Eventually AZA induced AQP1 ubiquitination, while proteasome inhibitor MG132 reversed AZA's down-regulating effect upon AQP1.Conclusions/significanceOur results identified AZA exerted diuretic effect through an innovative mechanism by regulating AQP1 and verified its inhibitory mechanism was via promoting MHC-dependent translocation onto cell membrane and then ubiquitin mediated degradation, implicating a novel mechanism and target for diuretic agent discovering.

Project description:BackgroundGlycerol outflow from adipocytes has been considered for a decade to be mediated by aquaporin-7, an aquaglyceroporin highly expressed in the adipose tissue. Its involvement in glycerol metabolism has been widely studied also in humans. Recent studies in different aquaporin-7 KO mice models pose two different questions 1) the exact localization of aquaporin-7 in human white adipose tissue; 2) the existence of other aquaglyceroporins that work with aquaporin-7 to guarantee glycerol efflux and thus a normal adiposity in humans. To this purpose we investigated the expression, the localization and the functioning of aquaglyceroporin-10 in subcutaneous white adipose tissue, in isolated and cultured differentiated adipocytes.Methodology/principal findingsAquaporin-7 and -10 were expressed in the white adipose tissue both at mRNA and at protein level. Immunofluorescence revealed aquaporin-7 and -10 labelling in the human adipose tissue both to the plasma membrane and to a thin rim of cytoplasm of adipocytes. Aquaporin-7, but not aquaporin-10, colocalized with the endothelial marker CD34. Human cultured differentiated adipocytes showed an aquaporin-7 and -10 labelling mainly in the cytoplasm and in the lipid droplets with insulin reinforcing the lipid droplets staining and isoproterenol inducing its translocation to the plasma membrane compartment. Water and glycerol permeability measurements using adipocytes and adipose membrane vesicles confirmed the presence of functioning aquaglyceroporins. Aquaporin-10 silencing in human differentiated adipocytes resulted in a 50% decrease of glycerol and osmotic water permeability.Conclusions/significanceThe results indicate that aquaporin-7, differently from mice, is present in both adipocyte and capillary plasma membranes of human adipose tissue. Aquaporin-10, on the contrary, is expressed exclusively in the adipocytes. The expression of two aquaglyceroporins in human adipose tissue is particularly important for the maintenance of normal or low glycerol contents inside the adipocyte, thus protecting humans from obesity.

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Coupled Mutations-Enabled Glycerol Transportation in an Aquaporin Z Mutant.

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Coupled Mutations-Enabled Glycerol Transportation in an Aquaporin Z Mutant.

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