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.

Protein aggregation is the etiopathogenesis of the three most profound vision-threatening eye diseases: age-related cataract, presbyopia, and age-related macular degeneration. This perspective organizes known information on ATP and protein aggregation with a fundamental unrecognized function of ATP. With recognition that maintenance of protein solubility is related to the high intracellular concentration of ATP in cells, tissues, and organs, we hypothesize that (1) ATP serves a critical molecular function for organismal homeostasis of proteins and (2) the hydrotropic feature of ATP prevents pathological protein aggregation while assisting in the maintenance of protein solubility and cellular, tissue, and organismal function. As such, the metabolite ATP plays an extraordinarily important role in the prevention of protein aggregation in the leading causes of vision loss or blindness worldwide.

Advanced glycation end products (AGEs) in lens proteins increase with aging, thus inducing cataracts and/or presbyopia. Hesperetin (Hst), which is an abundant plant flavanone largely derived from citrus species, and its derivatives attenuate cataracts and presbyopia in vivo and in vitro; however, no reports have described its effects on AGE formation in lens proteins. The present study demonstrated that AGEs in lens proteins increase with age in mice. Additionally, it showed that Hst can prevent AGEs and N(ε)‑carboxymethyl‑lysine generation and modification of lens proteins using in vitro in human lens epithelial cell lines and ex vivo in mouse lens organ cultures. Furthermore, treatment with Hst prevented lens hardening and decreased chaperone activity in lens proteins. These results suggested that Hst and its derivatives are good candidates for the prevention of presbyopia and cataracts.

Imaging mass spectrometry (IMS) enables targeted and untargeted visualization of the spatial localization of molecules in tissues with great specificity. The lens is a unique tissue that contains fiber cells corresponding to various stages of differentiation that are packed in a highly spatial order. The application of IMS to lens tissue localizes molecular features that are spatially related to the fiber cell organization. Such spatially resolved molecular information assists our understanding of lens structure and physiology; however, protein IMS studies are typically limited to abundant, soluble, low molecular weight proteins. In this study, a method was developed for imaging low solubility cytoskeletal proteins in the lens; a tissue that is filled with high concentrations of soluble crystallins. Optimized tissue washes combined with on-tissue enzymatic digestion allowed successful imaging of peptides corresponding to known lens cytoskeletal proteins. The resulting peptide signals facilitated segmentation of the bovine lens into molecularly distinct regions. A sharp intermediate filament transition from vimentin to lens-specific beaded filament proteins was detected in the lens cortex. MALDI IMS also revealed the region where posttranslational myristoylation of filensin occurs and the results indicate that truncation and myristoylation of filensin starts soon after filensin expression increased in the inner cortex. From intermediate filament switch to filensin truncation and myristoylation, multiple remarkable changes occur in the narrow region of lens cortex. MALDI images delineated the boundaries of distinct lens regions that will guide further proteomic and interactomic studies.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein-cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin-γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens-epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone-client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone-client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin-client complexes could contribute to lens aging and presbyopia.