AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency.
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ABSTRACT:
SUBMITTER: Gold MG
PROVIDER: S-EPMC3272850 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PurposeAquaporin 0 (AQP0) is the major intrinsic protein in the lens and is essential for establishing proper fiber cell structure and organization. Cytoskeletal proteins that directly interact with the C terminus of AQP0 are identified herein.MethodsThe water-insoluble fraction of lens fiber cells was chemically cross-linked, and cross-linked peptides with the C terminus of AQP0 were identified by mass spectrometry. Coimmunoprecipitation and AQP0 C-terminal peptide pulldown experiments were used to confirm the protein-protein interaction.ResultsUnexpectedly, AQP0 was found to directly associate with ezrin/radixin/moesin (ERM) family members, proteins that are involved in linkage of actin filaments to the plasma membrane. Cross-linked peptides were detected between AQP0 and degenerate sequences of ezrin and radixin; however, AQP0 interaction with ezrin is believed to play a more significant function in the lens because of its higher level of expression and observed ezrin-specific cross-linking. The interaction was found to occur between the C terminus of AQP0 and subdomains F1 and F3 of ERM proteins. The interaction between AQP0 and ezrin was confirmed by coimmunoprecipitation and AQP0 C-terminal peptide pulldown experiments.ConclusionsConsidering the important known functions of the cellular actin cytoskeleton in fiber cell differentiation, the interaction of AQP0 and ERM proteins may play an important role in fiber cell morphology, elongation, and organization.
| S-EPMC3176042 | biostudies-literature
Project description:Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.
| S-EPMC4461499 | biostudies-literature
Project description:Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.
| S-EPMC3831505 | biostudies-literature
Project description:Until recently, the lens was thought to express only two aquaporin (AQP) water channels, AQP1 and AQP0. In this study we confirm lenticular AQP5 protein expression by Western blotting and mass spectrometry in lenses from a variety of species. In addition, confocal microscopy was used to map cellular distributions of AQP5 in mouse, rat and human lenses. Tandem mass spectrometry of a human lens membrane preparation revealed extensive sequence coverage (56.2%) of AQP5. Western blotting performed on total fiber cell membranes from mouse, rat, bovine and human lenses confirmed AQP5 protein expression is conserved amongst species. Western blotting of dissected lens fractions suggests that AQP5 is processed in the lens core by C-terminal truncation. Immunohistochemistry showed that AQP5 signal was most abundant in the lens outer cortex and decreased in intensity in the lens core. Furthermore, AQP5 undergoes differentiation-dependent changes in subcellular location from an intracellular localization in differentiating fiber cells to the plasma membrane of mature fiber cells upon the loss of fiber cell nuclei. Our results show that AQP5 is a significant component of lens fiber cell membranes, representing the second most abundant water channel in these cells. Together, the changes to AQP5 distribution and structure are likely to modulate the functional role of AQP5 in different regions of the lens.
| S-EPMC3583522 | biostudies-literature
Project description:Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in ?-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3?, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial role for LTCCs in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the mechanical properties of lens, all of which have a vital role in maintaining lens function and cytoarchitecture.
| S-EPMC3667166 | biostudies-literature
Project description:The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.
| S-EPMC3117865 | biostudies-literature
Project description:An interaction between the C-terminus of aquaporin-0 (AQP0) and lens beaded filament protein filensin has been reported previously; however, the region of filensin that is involved in the interaction has not been determined. This study is designed to identify the region of filensin that interacts with AQP0. Chemical crosslinking coupled with mass spectrometry was used to identify the site of interaction. The protein complex was crosslinked with zero-length crosslinker: 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC). The crosslinked membrane fraction was digested by trypsin and crosslinked peptides were identified by liquid chromatography-tandem mass spectrometry. A crosslinked peptide between bovine filensin 450-465 (VKGPKEPEPPADLYTK) and bovine AQP0 239-259 (GSRPSESNGQPEVTGEPVELK) was detected. AQP0/filensin crosslinking was not detected in superficial young fiber cells, but increased with fiber cell age in the lens cortex. AQP0/filensin crosslinking and filensin truncation were observed in the same regions of the lens. This crosslinked peptide can be detected in 75 kDa gel band confirming that AQP0/filensin crosslinking can occur between AQP0 and the filensin C-terminal fragment. These results suggest that the AQP0 C-terminus directly interacts with the region of filensin that is adjacent to the major truncation site and the polybasic cluster of residues in the filensin C-terminal tail. This interaction occurs in a specific region of the lens and could only occur between AQP0 and filensin C-terminal fragment in vivo. This interaction supports the dual roles of filensin in the lens; roles that could be important during lens development.
| S-EPMC5471609 | biostudies-literature
Project description:Cell-to-cell adhesion (CTCA), which is key for establishing lens transparency, is a critical function of Aquaporin 0 (AQP0). The aim of this investigation was to find out the possible mechanism by which AQP0 exerts CTCA between fiber cells, since there are two proposals currently, either an AQP0-AQP0 interaction or an AQP0-lipid interaction. We studied the mechanism of AQP0-induced CTCA in intact AQP0 and C-terminally cleaved AQP0 (CTC-AQP0). Assays showed CTCA between L-cells transfected with intact AQP0 or CTC-AQP0 and parental L-cells indicating AQP0-membrane interaction. Both forms of AQP0 significantly (P?<?0.001) promoted adhesion to negatively charged l-?-phosphatidylserine lipid vesicles signifying AQP0-lipid interaction. AQP0-expressing L-cells also promoted adhesion of WT and AQP0-KO mouse lens fiber cell membrane vesicles (FCMVs) significantly (P?<?0.001). However, when FCMVs of WT or AQP0-KO were plated over parental L-cells, only WT vesicles adhered significantly, corroborating AQP0-membrane interaction. After incubating with extracellular domain-specific AQP0 antibody, L-cells expressing intact AQP0 or CTC-AQP0 showed a significant reduction (P?<?0.001) in the adhesion of AQP0-KO FCMVs indicating extracellular loop involvement in CTCA. WT FCMVs from outer cortex and inner cortex promoted adhesion to parental L-cells, without any statistically significant difference in adhesion efficiency (P?>?0.05). Ultrastructure studies of WT, AQP0-KO and transgenic lenses showed AQP0 is critical for fiber CTCA and compaction. The data collected clearly demonstrate that the positively charged amino acids in the AQP0 extracellular loop domains interact with the negatively charged lipids in the plasma membrane to promote CTCA for compaction of fiber cells.
| S-EPMC6223623 | biostudies-literature
Project description:Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 ?m. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation.
| S-EPMC3267704 | biostudies-literature
Project description:PurposeTo spatially map human lens Aquaporin-0 (AQP0) protein modifications, including lipidation, truncation, and deamidation, from birth through middle age using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS).MethodsHuman lens sections were water-washed to facilitate detection of membrane protein AQP0. We acquired MALDI images from eight human lenses ranging in age from 2 months to 63 years. In situ tryptic digestion was used to generate peptides of AQP0 and peptide images were acquired on a 15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Peptide extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searched to identify peptides observed in MALDI imaging experiments.ResultsUnmodified, truncated, and fatty acid-acylated forms of AQP0 were detected in protein imaging experiments. Full-length AQP0 was fatty acid acylated in the core and cortex of young (2- and 4-month) lenses. Acylated and unmodified AQP0 were C-terminally truncated in older lens cores. Deamidated tryptic peptides (+0.9847 Da) were mass resolved from unmodified peptides by FTICR MS. Peptide images revealed differential localization of un-, singly-, and doubly-deamidated AQP0 C-terminal peptide (239-263). Deamidation was present at 4 months and increases with age. Liquid chromatography-MS/MS results indicated N246 undergoes deamidation more rapidly than N259.ConclusionsResults indicated AQP0 fatty acid acylation and deamidation occur during early development. Progressive age-related AQP0 processing, including deamidation and truncation, was mapped in human lenses as a function of age. The localization of these modified AQP0 forms suggests where AQP0 functions may change throughout lens development and aging.
| S-EPMC4675204 | biostudies-literature