The role of Aquaporins in tumorigenesis: implications for therapeutic development.

Aquaporins (AQPs) are ubiquitous channel proteins that play a critical role in the homeostasis of the cellular environment by allowing the transit of water, chemicals, and ions. They can be found in many different types of cells and organs, including the lungs, eyes, brain, glands, and blood vessels. By controlling the osmotic water flux in processes like cell growth, energy metabolism, migration, adhesion, and proliferation, AQPs are capable of exerting their regulatory influence over a wide range of cellular processes. Tumour cells of varying sources express AQPs significantly, especially in malignant tumours with a high propensity for metastasis. New insights into the roles of AQPs in cell migration and proliferation reinforce the notion that AQPs are crucial players in tumour biology. AQPs have recently been shown to be a powerful tool in the fight against pathogenic antibodies and metastatic cell migration, despite the fact that the molecular processes of aquaporins in pathology are not entirely established. In this review, we shall discuss the several ways in which AQPs are expressed in the body, the unique roles they play in tumorigenesis, and the novel therapeutic approaches that could be adopted to treat carcinoma.

Aquaporin water channels: roles beyond renal water handling.

Aquaporin (AQP) water channels are pivotal to renal water handling and therefore in the regulation of body water homeostasis. However, beyond the kidney, AQPs facilitate water reabsorption and secretion in other cells and tissues, including sweat and salivary glands and the gastrointestinal tract. A growing body of evidence has also revealed that AQPs not only facilitate the transport of water but also the transport of several small molecules and gases such as glycerol, H2O2, ions and CO2. Moreover, AQPs are increasingly understood to contribute to various cellular processes, including cellular migration, adhesion and polarity, and to act upstream of several intracellular and intercellular signalling pathways to regulate processes such as cell proliferation, apoptosis and cell invasiveness. Of note, several AQPs are highly expressed in multiple cancers, where their expression can correlate with the spread of cancerous cells to lymph nodes and alter the response of cancers to conventional chemotherapeutics. These data suggest that AQPs have diverse roles in various homeostatic and physiological systems and may be exploited for prognostics and therapeutic interventions.

Adaptive Selection in the Evolution of Aquaglyceroporins in Mammals.

Aquaporins (AQPs) are integral membrane proteins responsible for water transport across cellular membranes in both prokaryotes and eukaryotes. A subfamily of AQPs, known as aquaglyceroporins (AQGPs), facilitate the transport of small solutes such as glycerol, water, and other solutes across cellular membranes. These proteins are involved in a variety of physiological processes, such as organogenesis, wound healing, and hydration. Although AQPs have been studied extensively in different species, their conservation patterns, phylogenetic relationships, and evolution in mammals remain unexplored. In the present study, 119 AQGP coding sequences from 31 mammalian species were analysed to identify conserved residues, gene organisation, and most importantly, the nature of AQGP gene selection. Repertoire analysis revealed the absence of AQP7, 9, and 10 genes in certain species of Primates, Rodentia, and Diprotodontia, although not all three genes were absent in a single species. Two Asparagine-Proline-Alanine (NPA) motifs located at the N- and C-terminal ends, aspartic acid (D) residues, and the ar/R region were conserved in AQP3, 9, and 10. Six exons encoding the functional MIP domain of AQGP genes were found to be conserved across mammalian species. Evolutionary analysis indicated signatures of positive selection in AQP7, 9, and 10 amongst different mammalian lineages. Furthermore, substitutions of certain amino acids located close to critical residues may alter AQGP functionality, which is crucial for substrate selectivity, pore formation, and transport efficiency required for the maintenance of homeostasis in different mammalian species.

Unraveling the Aquaporin-3 Inhibitory Effect of Rottlerin by Experimental and Computational Approaches.

The natural polyphenolic compound Rottlerin (RoT) showed anticancer properties in a variety of human cancers through the inhibition of several target molecules implicated in tumorigenesis, revealing its potential as an anticancer agent. Aquaporins (AQPs) are found overexpressed in different types of cancers and have recently emerged as promising pharmacological targets. Increasing evidence suggests that the water/glycerol channel aquaporin-3 (AQP3) plays a key role in cancer and metastasis. Here, we report the ability of RoT to inhibit human AQP3 activity with an IC50 in the micromolar range (22.8 ± 5.82 µM for water and 6.7 ± 2.97 µM for glycerol permeability inhibition). Moreover, we have used molecular docking and molecular dynamics simulations to understand the structural determinants of RoT that explain its ability to inhibit AQP3. Our results show that RoT blocks AQP3-glycerol permeation by establishing strong and stable interactions at the extracellular region of AQP3 pores interacting with residues essential for glycerol permeation. Altogether, our multidisciplinary approach unveiled RoT as an anticancer drug against tumors where AQP3 is highly expressed providing new information to aquaporin research that may boost future drug design.

dbAQP-SNP: a database of missense single-nucleotide polymorphisms in human aquaporins.

Aquaporins and aquaglyceroporins belong to the superfamily of major intrinsic proteins (MIPs), and they transport water and other neutral solutes such as glycerol. These channel proteins are involved in vital physiological processes and are implicated in several human diseases. Experimentally determined structures of MIPs from diverse organisms reveal a unique hour-glass fold with six transmembrane helices and two half-helices. MIP channels have two constrictions formed by Asn-Pro-Ala (NPA) motifs and aromatic/arginine selectivity filters (Ar/R SFs). Several reports have found associations among single-nucleotide polymorphisms (SNPs) in human aquaporins (AQPs) with diseases in specific populations. In this study, we have compiled 2798 SNPs that give rise to missense mutations in 13 human AQPs. To understand the nature of missense substitutions, we have systematically analyzed the pattern of substitutions. We found several examples in which substitutions could be considered as non-conservative that include small to big or hydrophobic to charged residues. We also analyzed these substitutions in the context of structure. We have identified SNPs that occur in NPA motifs or Ar/R SFs, and they will most certainly disrupt the structure and/or transport properties of human AQPs. We found 22 examples in which missense SNP substitutions that are mostly non-conservative in nature have given rise to pathogenic conditions as found in the Online Mendelian Inheritance in Man database. It is most likely that not all missense SNPs in human AQPs will result in diseases. However, understanding the effect of missense SNPs on the structure and function of human AQPs is important. In this direction, we have developed a database dbAQP-SNP that contains information about all 2798 SNPs. This database has several features and search options that can help the user to find SNPs in specific positions of human AQPs including the functionally and/or structurally important regions. dbAQP-SNP (http://bioinfo.iitk.ac.in/dbAQP-SNP) is freely available to the academic community. Database URL http://bioinfo.iitk.ac.in/dbAQP-SNP.

Molecular dynamics simulation and structural analysis of aquaporin Z from an Antarctic Pseudomonas sp. strain AMS3.

Aquaporin is a water channel protein that facilitates the movement of water across the cell membrane. Aquaporin from the Antarctic region has been noted for its psychrophilic properties and its ability to perform at a lower temperature but there remains limited understanding of the water mechanism of Antarctic Pseudomonas sp. strain AMS3 However, studies regarding aquaporin isolated from psychrophilic Pseudomonas sp. are still scattered. Recently, the genome sequence of an Antarctic Pseudomonas sp. strain AMS3 revealed a gene sequence encoding for a putative aquaporin designated as AqpZ1 AMS3. In this study, structure analysis and a molecular dynamics (MD) simulation of a predicted model of a fully hydrated aquaporin tetramer embedded in a lipid bilayer was performed at different temperatures for structural flexibility and stability analysis. The MD simulation results revealed that the structures were able to remain stable at low to medium temperatures. The protein was observed to have high flexibility in the loop region as compared to the helices region throughout the simulated temperatures. The selectivity filter and NPA motifs play a major role in solute selectivity and the pore radius of the protein. The structural and functional characterization of this psychrophilic aquaporin provides new insights for the future applications of this protein.Communicated by Ramaswamy H. Sarma.

Classification and Gene Structure of Aquaporins.

Aquaporins (AQPs) are a family of membrane water channels that basically function as regulators of intracellular and intercellular water flow. To date, 13 AQPs, distributed widely in specific cell types in various organs and tissues, have been characterized in humans. A pair of NPA boxes forming a pore is highly conserved among all aquaporins and is also key residues for the classification of AQP superfamily into four groups according to primary sequences. AQPs may also be classified based on their transport properties. So far, chromosome localization and gene structure of 13 human AQPs have been identified, which is definitely helpful for studying phenotypes and potential targets in naturally occurring and synthetic mutations in human or cells.

Protein Structure and Modification of Aquaporins.

Aquaporins (AQPs) allow water molecules and other small, neutral solutes to quickly pass through membrane. The protein structures of AQPs solved by crystallographic methods or cryo-electron microscopy technology show that AQP monomer consists of six membrane-spanning alpha-helices that form the central water-transporting pore. AQP monomers assemble to form tetramers, forming the functional units in the membrane, to transport water or other small molecules. The biological functions of AQPs are regulated by posttranslational modifications, e.g., phosphorylation, ubiquitination, glycosylation, subcellular distribution, degradation and protein interactions. Modifications of AQP combined with structural properties contribute to a better functional mechanism of AQPs. Insight into the molecular mechanisms responsible for AQP modifications as well as gating and transport properties proved to be fundamental to the development of new therapeutic targets or reliable diagnostic and prognostic biomarkers.

Evolutionary Overview of Aquaporin Superfamily.

Aquaporins (AQPs) are present not only in three domains of life, bacteria, eukaryotes, and archaea, but also in viruses. With the accumulating arrays of AQP superfamily, the evolutional relationship has attracted much attention with multiple publications on "the genome-wide identification and phylogenetic analysis" of AQP superfamily. A pair of NPA boxes forming a pore is highly conserved throughout the evolution and renders key residues for the classification of AQP superfamily into four groups: AQP1-like, AQP3-like, AQP8-like, and AQP11-like. The complexity of AQP family has mostly been achieved in nematodes and subsequent evolution has been directed toward increasing the number of AQPs through whole-genome duplications (WGDs) to extend the tissue specific expression and regulation. The discovery of the intracellular AQP (iAQP: AQP8-like and AQP11-like) and substrate transports by the plasma membrane AQP (pAQP: AQP1-like and AQP3-like) have accelerated the AQP research much more toward the transport of substrates with complex profiles. This evolutionary overview based on a simple classification of AQPs into four subfamilies will provide putative structural, functional, and localization information and insights into the role of AQP as well as clues to understand the complex diversity of AQP superfamily.

Aquaporins in Skin.

The skin is the largest organ of our body and plays a protective role against the external environment. The skin functions as a mechanical and water permeability barrier, assisting with thermoregulation and defending our body against a variety of stresses such as ultraviolet radiation, microbial infection, physical injuries, and chemical hazards. The structure of the skin consists of three main layers: the hypodermis, the dermis, and the epidermis. Aquaporins (AQPs) are a family of integral membrane proteins whose function is to regulate intracellular fluid hemostasis by facilitating the transportation of water, and in some cases small molecules, across the cell membranes. Up to six different AQPs (AQP1, 3, 5, 7, 9, and 10) are expressed in a variety of cell types in the skin. The AQP family plays an important role in these various locations, contributing to many key functions of the skin including hydration, wound healing, and immune responses. The involvement of different aquaporin family members in skin is discussed.

Coronatine promotes maize water uptake by directly binding to the aquaporin ZmPIP2;5 and enhancing its activity.

Water uptake is crucial for crop growth and development and drought stress tolerance. The water channel aquaporins (AQP) play important roles in plant water uptake. Here, we discovered that a jasmonic acid analog, coronatine (COR), enhanced maize (Zea mays) root water uptake capacity under artificial water deficiency conditions. COR treatment induced the expression of the AQP gene Plasma membrane intrinsic protein 2;5 (ZmPIP2;5). In vivo and in vitro experiments indicated that COR also directly acts on ZmPIP2;5 to improve water uptake in maize and Xenopus oocytes. The leaf water potential and hydraulic conductivity of roots growing under hyperosmotic conditions were higher in ZmPIP2;5-overexpression lines and lower in the zmpip2;5 knockout mutant, compared to wild-type plants. Based on a comparison between ZmPIP2;5 and other PIP2s, we predicted that COR may bind to the functional site in loop E of ZmPIP2;5. We confirmed this prediction by surface plasmon resonance technology and a microscale thermophoresis assay, and showed that deleting the binding motif greatly reduced COR binding. We identified the N241 residue as the COR-specific binding site, which may activate the channel of the AQP tetramer and increase water transport activity, which may facilitate water uptake under hyperosmotic stress.

Genome-wide identification and biological relevance of broccoli aquaporins.

Broccoli (Brassica oleracea var. italica) is an important crop worldwide, and its regular consumption is associated with health benefits due to the presence of various bioactive compounds. An optimal water balance and homeostasis are needed for plant growth; in this sense, aquaporins play a crucial role. As a result of a genome-wide search, a total of 65 aquaporin genes were identified in broccoli. The aquaporins were classified according to their phylogenetic relationships with other Brassicas species and Arabidopsis thaliana, and evolutionary events of gene duplication were also assessed, highlighting the tendency of NIPs (Nodulin-26-like Intrinsic Proteins) to duplicate. Also, the chromosomal localization, gene duplication, the study of the conserved motifs, and the tertiary structure were determined in broccoli. Functional predictive analyses were also carried out, which, together with the expression analyses in different broccoli plant tissues, allowed the prediction of the biological role of each aquaporin isoform. BoiPIP1-2a and BoiPIP1-2b showed higher expression in all the plant tissues when compared with other aquaporins. BoiTIP1-2b also showed high expression levels and was associated with nitrogen compounds transport such as urea. However, NIPs, through their differential expression and the tandem duplications of the isoforms, were revealed as the putative main actors in the response of broccoli plants to abiotic stress responses. The results of this work pointed to the physiological significance of each aquaporin isoform of broccoli, opening a new field of knowledge and constituting the first step of further in vivo analyses.

High-resolution structure of a fish aquaporin reveals a novel extracellular fold.

Aquaporins are protein channels embedded in the lipid bilayer in cells from all organisms on earth that are crucial for water homeostasis. In fish, aquaporins are believed to be important for osmoregulation; however, the molecular mechanism behind this is poorly understood. Here, we present the first structural and functional characterization of a fish aquaporin; cpAQP1aa from the fresh water fish climbing perch (<i>Anabas testudineus</i>), a species that is of high osmoregulatory interest because of its ability to spend time in seawater and on land. These studies show that cpAQP1aa is a water-specific aquaporin with a unique fold on the extracellular side that results in a constriction region. Functional analysis combined with molecular dynamic simulations suggests that phosphorylation at two sites causes structural perturbations in this region that may have implications for channel gating from the extracellular side.

Multiple pore lining residues modulate water permeability of GlpF.

The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin "open state" is still considered to be a continuously open pore with water molecules permeating in a single-file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long-term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications.

Beyond Aquaporins: Recent Developments in Artificial Water Channels.

A molecular scale understanding of the fast and selective water transport in biological water channels, aquaporins (AQPs), has inspired attempts to mimic its performance in synthetic structures. These synthetic structures, referred to as artificial water channels (AWCs), present several advantages over AQPs in applications. After over a decade of efforts, the unique transport properties of AQPs have been reproduced in AWCs. Further, recent developments have shown that the performance of benchmark AQP channels can be exceeded by new AWC designs using novel features not seen in biology. In this Perspective, we provide a brief overview of recent AWC developments, and share our perspective on forward-looking AWC research.

The Hidden Intricacies of Aquaporins: Remarkable Details in a Common Structural Scaffold.

Evolution turned aquaporins (AQPs) into the most efficient facilitators of passive water flow through cell membranes at no expense of solute discrimination. In spite of a plethora of solved AQP structures, many structural details remain hidden. Here, by combining extensive sequence- and structural-based analysis of a unique set of 20 non-redundant high-resolution structures and molecular dynamics simulations of four representatives, key aspects of AQP stability, gating, selectivity, pore geometry, and oligomerization, with a potential impact on channel functionality, are identified. The general view of AQPs possessing a continuous open water pore is challenged and it is depicted that AQPs' selectivity is not exclusively shaped by pore-lining residues but also by the relative arrangement of transmembrane helices. Moreover, this analysis reveals that hydrophobic interactions constitute the main determinant of protein thermal stability. Finally, a numbering scheme of the conserved AQP scaffold is established, facilitating direct comparison of, for example, disease-causing mutations and prediction of potential structural consequences. Additionally, the results pave the way for the design of optimized AQP water channels to be utilized in biotechnological applications.

Fluorofoldamer-Based Salt- and Proton-Rejecting Artificial Water Channels for Ultrafast Water Transport.

Here, we report on a novel class of fluorofoldamer-based artificial water channels (AWCs) that combines excellent water transport rate and selectivity with structural simplicity and robustness. Produced by a facile one-pot copolymerization reaction under mild conditions, the best-performing channel (AWC 1) is an n-C8H17-decorated foldamer nanotube with an average channel length of 2.8 nm and a pore diameter of 5.2 Å. AWC 1 demonstrates an ultrafast water conduction rate of 1.4 × 1010 H2O/s per channel, outperforming the archetypal biological water channel, aquaporin 1, while excluding salts (i.e., NaCl and KCl) and protons. Unique to this class of channels, the inwardly facing C(sp2)-F atoms being the most electronegative in the periodic table are proposed as being critical to enabling the ultrafast and superselective water transport properties by decreasing the channel's cavity and enhancing the channel wall smoothness via reducing intermolecular forces with water molecules or hydrated ions.

Beating natural proteins at filtering water.

Artificial fluorous channels outperform aquaporins in water permeation.

Ultrafast water permeation through nanochannels with a densely fluorous interior surface.

Ultrafast water permeation in aquaporins is promoted by their hydrophobic interior surface. Polytetrafluoroethylene has a dense fluorine surface, leading to its strong water repellence. We report a series of fluorous oligoamide nanorings with interior diameters ranging from 0.9 to 1.9 nanometers. These nanorings undergo supramolecular polymerization in phospholipid bilayer membranes to form fluorous nanochannels, the interior walls of which are densely covered with fluorine atoms. The nanochannel with the smallest diameter exhibits a water permeation flux that is two orders of magnitude greater than those of aquaporins and carbon nanotubes. The proposed nanochannel exhibits negligible chloride ion (Cl-) permeability caused by a powerful electrostatic barrier provided by the electrostatically negative fluorous interior surface. Thus, this nanochannel is expected to show nearly perfect salt reflectance for desalination.

Investigating the Lipid Selectivity of Membrane Proteins in Heterogeneous Nanodiscs.

The structure and function of membrane proteins can be significantly impacted by the surrounding lipid environment, but membrane protein-lipid interactions in lipid bilayers are often difficult to study due to their transient and polydisperse nature. Here, we used two native mass spectrometry (MS) approaches to investigate how the Escherichia coli ammonium transporter trimer (AmtB) and aquaporin Z (AqpZ) selectively remodel their local lipid environment in heterogeneous lipoprotein nanodiscs. First, we used gas-phase ejection to isolate the membrane protein with bound lipids from heterogeneous nanodiscs with different combinations of lipids. Second, we used solution-phase detergent extraction as an orthogonal approach to study membrane protein remodeling of lipids in the nanodisc with native MS. Our results showed that Triton X-100 and lauryldimethylamine oxide retain lipid selectivity that agrees with gas-phase ejection, but C8E4 distorts some preferential lipid interactions. Both approaches reveal that AmtB has a few selective binding sites for phosphatidylcholine (PC) lipids, is selective for binding phosphatidylglycerols (PG) overall, and is nonselective for phosphatidylethanolamines (PE). In contrast, AqpZ prefers either PC or PG over PE and prefers PC over PG. Overall, these experiments provide a picture of how membrane proteins bind different lipid head groups in the context of mixed lipid bilayers.