Aquaporin-0-protein interactions elucidated by crosslinking mass spectrometry.

Aquaporin-0 (AQP0) constitutes 50 % of the lens membrane proteome and plays important roles in lens fiber cell adhesion, water permeability, and lens transparency. Previous work has shown that specific proteins, such as calmodulin (CaM), interact with AQP0 to modulate its water permeability; however, these studies often used AQP0 peptides, rather than full-length protein, to probe these interactions. Furthermore, the specific regions of interaction of several known AQP0 interacting partners, i.e. αA and αB-crystallins, and phakinin (CP49) remain unknown. The purpose of this study was to use crosslinking mass spectrometry (XL-MS) to identify interacting proteins with full-length AQP0 in crude lens cortical membrane fractions and to determine the specific protein regions of interaction. Our results demonstrate, for the first time, that the AQP0 N-terminus can engage in protein interactions. Specific regions of interaction are elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50. In addition, two new interacting partners, vimentin and connexin-46, were identified.

Insight into the Mammalian Aquaporin Interactome.

Aquaporins (AQPs) are a family of transmembrane water channels expressed in all living organisms. AQPs facilitate osmotically driven water flux across biological membranes and, in some cases, the movement of small molecules (such as glycerol, urea, CO2, NH3, H2O2). Protein-protein interactions play essential roles in protein regulation and function. This review provides a comprehensive overview of the current knowledge of the AQP interactomes and addresses the molecular basis and functional significance of these protein-protein interactions in health and diseases. Targeting AQP interactomes may offer new therapeutic avenues as targeting individual AQPs remains challenging despite intense efforts.

Lens Aquaporins in Health and Disease: Location is Everything!

Cataract and presbyopia are the leading cause of vision loss and impaired vision, respectively, worldwide. Changes in lens biochemistry and physiology with age are responsible for vision impairment, yet the specific molecular changes that underpin such changes are not entirely understood. In order to preserve transparency over decades of life, the lens establishes and maintains a microcirculation system (MCS) that, through spatially localized ion pumps, induces circulation of water and nutrients into (influx) and metabolites out of (outflow and efflux) the lens. Aquaporins (AQPs) are predicted to play important roles in the establishment and maintenance of local and global water flow throughout the lens. This review discusses the structure and function of lens AQPs and, importantly, their spatial localization that is likely key to proper water flow through the MCS. Moreover, age-related changes are detailed and their predicted effects on the MCS are discussed leading to an updated MCS model. Lastly, the potential therapeutic targeting of AQPs for prevention or treatment of cataract and presbyopia is discussed.

Native Mass Spectrometry and Surface Induced Dissociation Provide Insight into the Post-Translational Modifications of Tetrameric AQP0 Isolated from Bovine Eye Lens.

Aquaporin-0 (AQP0) is a tetrameric membrane protein and the most abundant membrane protein in the eye lens. Interestingly, there is little to no cellular turnover once mature lens fiber cells are formed, and hence, age-related modifications accumulate with time. While bottom-up mass spectrometry-based approaches can provide identification of post-translational modifications, they cannot provide information on how these modifications coexist in a single chain or complex. Native mass spectrometry, however, enables the transfer of the intact complex into the gas-phase allowing modifications to be identified at the tetramer level. Here, we present the use of native mass spectrometry and surface-induced dissociation to study the post-translational modifications of AQP0 isolated and purified from bovine eye lens, existing as multiple forms due to the different modification states naturally present.