Biology subjects

Schey, K. L.

Publications and source records attributed to Schey, K. L..

12 recordsLinked to original sources

bioRxiv2026

Upregulation of the Unfolded and Mitochondrial Unfolded Protein Responses in Oxidative Stress-Induced Cataract

Purpose: Oxidative stress is thought to contribute to the development of age-related cataracts (ARCs), but the mechanisms by which oxidative damage leads to the opacification of the lens remain unclear. Previous studies suggest that oxidative stress can disrupt lens proteostasis. Therefore, it was hypothesized that ARCs arise from proteomic changes driven by an age-associated decline in oxidative stress defenses that interact with the lens proteostatic state. To test this hypothesis, proteomic analyses of lenses exposed to oxidative stress were performed to examine oxidative and proteostatic stress responses in vivo. Methods: Cataract formation was induced by injecting hydrogen peroxide into the aqueous humor of adult zebrafish. nrf2fh318/fh318 zebrafish were used to model the reduced oxidative stress protection observed in aged human lenses, while cryaba-/- zebrafish were used to model impaired lens proteostasis. Resulting opacities in WT, cryaba-/-, nrf2fh318/fh318, and cryaba-/-; nrf2fh318/fh318 lenses were quantified and proteomic changes in the cortex were analyzed using data independent acquisition Parallel Accumulation Serial Fragmentation mass spectrometry. Results: Hydrogen peroxide treatment induced the formation of cortical cataracts. Proteomic results showed that, dependent on genotype and day, oxidative stress activates the unfolded and mitochondrial unfolded protein responses. Additional changes were also observed in energy metabolism, Ca2+ homeostasis, protein degradation, and cytoskeletal and extracellular matrix remodeling pathways. Conclusions: Treated zebrafish lenses successfully model ARC and mass spectrometry proteomics identified the unfolded and mitochondrial unfolded protein responses as potential therapeutic targets for ARC.

molecular biology↗

bioRxiv2026

PEDF/PEDF-R Signaling Regulates Retinal Phospholipid Homeostasis and Photoreceptor Survival

Pigment epithelium-derived factor (PEDF) promotes photoreceptor survival through its receptor PEDF-R, a phospholipase involved in retinal lipid metabolism. To define the in vivo function of the PEDF/PEDF-R axis, we generated mice lacking Serpinf1 (PEDF) and Pnpla2 (PEDF-R). Combined loss of Serpinf1 and Pnpla2 resulted in severe retinal degeneration characterized by outer nuclear layer (ONL) thinning, outer segment (OS) shortening, reduced rhodopsin and cone opsin expression, increased TUNEL-positive nuclei, and enhanced retinal autofluorescence associated with altered lipid distribution. Lipid-associated markers, including TIP47, PLIN5, and BODIPY, exhibited abnormal distribution patterns in mutant retinas, indicating disrupted lipid storage and trafficking. Loss of PEDF/PEDF-R signaling also impaired photoreceptor-rod bipolar cell connectivity, as demonstrated by reduced PKC/synaptophysin colocalization, and resulted in diminished electroretinographic responses. Lipid Imaging mass spectrometry revealed decreases in some lipid abundances in photoreceptor outer segment and inner segment/outer nucleus layer, while lipids containing arachidonic acid and docosahexaenoic acid-containing lipids showed increased abundance. Together these findings identify the PEDF/PEDF-R signaling axis as a key regulator of retinal phospholipid homeostasis that couples lipid metabolism to photoreceptor survival and visual function.

neuroscience↗

bioRxiv2025

Data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) analysis of the separated zebrafish lens improves identifications

Ocular lens fiber cells degrade their organelles during differentiation to prevent light scattering. Organelle degradation occurs continuously throughout an individuals lifespan, creating a spatial gradient of young cortical fiber cells in the lens periphery to older nuclear fiber cells in the center of the lens. Therefore, separation of cortical and nuclear regions enables examination of protein aging. Previously, the human lens cortex and nucleus have been studied using data-independent acquisition (DIA) proteomics, allowing for the identification of low-abundance protein groups. In this study, we employed data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) proteomics on a timsTOF HT instrument to study the zebrafish lens proteome and compared results to a standard DIA method employed on an Orbitrap Exploris 480. Using the additional ion mobility gas phase separation of diaPASEF, peptide and protein group identifications increased by over 200% relative to an orbitrap DIA method in the zebrafish lens. With diaPASEF, we identified 13,721 and 11,996 unique peptides in the zebrafish lens cortex and nucleus, respectively, which correspond to 1,537 and 1,389 protein groups. Thus, separation of the zebrafish lens into cortical and nuclear regions followed by diaPASEF analysis produced the most comprehensive zebrafish lens proteomic dataset to date.

molecular biology↗

bioRxiv2022

Proteome Remodeling of the Eye Lens at 50 Years Identified with Data-Independent Acquisition

The eye lens is responsible for focusing and transmitting light to the retina. The lens does this in the absence of organelles yet maintains transparency for at least five decades before onset of age-related nuclear cataract (ARNC). It is hypothesized that oxidative stress contributes significantly to ARNC formation. It is additionally hypothesized that transparency is maintained by a microcirculation system (MCS) that delivers antioxidants to the lens nucleus and exports small molecule waste. Common data-ependent acquisition (DDA) methods are hindered by dynamic range of lens protein expression and provide limited context to age-related changes in the lens. In this study we utilized data-independent acquisition (DIA) mass spectrometry to analyze the urea insoluble, membrane protein fractions of 16 human lenses subdivided into three spatially distinct lens regions to characterize age-related changes, particularly concerning the lens MCS and oxidative stress response. In this pilot cohort, we measured 4,788 distinct protein groups, 46,681 peptides, and 7,592 deamidated sequences, more than in any previous human lens DDA approach. Our results reveal age-related changes previously known in lens biology and expand on these findings, taking advantage of the rich dataset afforded by DIA. Principally, we demonstrate that a significant proteome remodeling event occurs at approximately 50 years of age, resulting in metabolic preference for anaerobic glycolysis established with organelle degradation, decreased abundance of protein networks involved in calcium-dependent cell-cell contacts while retaining networks related to oxidative stress response. Further, we identified multiple antioxidant transporter proteins not previously detected in the human lens and describe their spatiotemporal and age-related abundance changes. Finally, we demonstrate that aquaporin-5, among other proteins, is modified with age by PTMs including deamidation and truncation. We suggest that the continued accumulation of each of these age-related outcomes in proteome remodeling contribute to decreased fiber cell permeability and result in ARNC formation.

biochemistry↗

bioRxiv2022

Lens aquaporin-5 inserts into bovine fiber cell plasma membranes through mitochondria-associated lysosome secretion

PURPOSETo spatially map aquaporin-5 (AQP5) expression in bovine lens, molecularly characterize cytoplasmic AQP5-containing vesicles in the outer cortex, and elucidate AQP5 membrane trafficking mechanisms. METHODSImmunofluorescence was performed on bovine lens cryosections using AQP5, TOMM20, COX IV, calnexin, LC3B, LIMP-2, and connexin-50 antibodies and the fluorescent lipid membrane dye CM-DiI. AQP5 plasma membrane insertion was defined via line expression profile analysis. Transmission electron microscopy (TEM) was performed on bovine lens tissue sections to define cytoplasmic organelle identity, morphology, and subcellular localization in cortical fiber cells. Bovine lenses were treated with 10 nM bafilomycin A1 or 0.1% dimethyl sulfoxide vehicle control in ex vivo culture to determine changes in AQP5 plasma membrane expression. RESULTSImmunofluorescence analysis revealed cytoplasmic AQP5 expression in bovine lens epithelial cells and differentiating fiber cells. In the bovine lens cortex, complete AQP5 plasma membrane insertion occurs at r/a 0.951 + 0.005. AQP5-containing cytoplasmic vesicles are spheroidal, tubular in morphology, express TOMM20, and contain LC3B and LIMP-2 as fiber cells mature. TEM analysis revealed spheroidal, tubular autophagosomes, autolysosomes, and lysosomes with degrading mitochondria. AQP5-containing cytoplasmic vesicles and autolysosomes dock and fuse with the plasma membrane. Bafiloymcin A1 treatment reduced AQP5 plasma membrane expression by 27%. CONCLUSIONSAQP5 localizes to spheroidal, tubular cytoplasmic vesicles in the differentiating bovine lens fiber cells. During fiber cell differentiation, these vesicles incorporate LC3B and fuse with LIMP-2-positive lysosomes. AQP5 trafficking to the plasma membrane occurs through lysosome secretion as a novel mechanism of AQP5 trafficking.

cell biology↗

bioRxiv2021

Data Independent Acquisition Mass Spectrometry of the Human Lens Enhances Spatiotemporal Measurement of Fiber Cell Aging

The ocular lens proteome undergoes post-translational and progressive degradation as fiber cells age. The oldest fiber cells and the proteins therein are present at birth and are retained through death. Transparency of the lens is maintained in part by the high abundance crystallin family proteins (up to 300 mg/mL), which establishes a high dynamic range of protein abundance. As a result, previous Data Dependent Analysis (DDA) measurements of the lens proteome are less equipped to identify the lowest abundance proteins. In an attempt to probe more deeply into the lens proteome, we measured the insoluble lens proteome of an 18-year-old human with DDA and newer Data Independent Analysis (DIA) methods. By applying library free DIA search methods, 4,564 protein groups, 48,474 peptides and 5,577 deamidation sites were detected: significantly outperforming the quantity of identifications in using DDA and Pan-Human DIA library searches. Finally, by segmenting the lens into multiple fiber cell-age related regions, we uncovered cell-age resolved changes in proteome composition and putative function.

biochemistry↗

bioRxiv2021

Tissue fixation effects on human retinal lipid analysis by MALDI imaging and LC-MS/MS technologies

Imaging mass spectrometry (IMS) allows the location and abundance of lipids to be mapped across tissue sections of human retina. For reproducible and accurate information, sample preparation methods need to be optimized. Paraformaldehyde fixation of a delicate multilayer structure like human retina facilitates the preservation of tissue morphology by forming methylene bridge cross-links between formaldehyde and amine/ thiols in biomolecules; however, retina sections analyzed by IMS are typically fresh-frozen. To determine if clinically significant inferences could be reliably based on fixed tissue, we evaluated the effect of fixation on analyte detection, spatial localization, and introduction of artefactual signals. Hence, we assessed the molecular identity of lipids generated by matrix-assisted laser desorption ionization (MALDI-IMS) and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) for fixed and fresh-frozen retina tissues in positive and negative ion modes. Based on MALDI-IMS analysis, more lipid signals were observed in fixed compared to fresh-frozen retina. More potassium adducts were observed in fresh-frozen tissues than fixed as the fixation process caused displacement of potassium adducts to protonated and sodiated species in ion positive ion mode. LC-MS/MS analysis revealed an overall decrease in lipid signals due to fixation that reduced glycerophospholipids and glycerolipids and conserved most sphingolipids and cholesteryl esters. The high quality and reproducible information from untargeted lipidomics analysis of fixed retina informs on all major lipid classes, similar to fresh-frozen retina, and serves as a steppingstone towards understanding of lipid alterations in retinal diseases.

biochemistry↗

bioRxiv2021

Spatially-Resolved Proteomic Analysis of the Lens Extracellular Diffusion Barrier

PurposeThe presence of a physical barrier to molecular diffusion through lenticular extracellular space has been repeatedly detected in multiple species. This extracellular diffusion barrier has been proposed to restrict the movement of solutes into the lens and to direct nutrients into the lens core via the sutures at both poles. The purpose of this study is to characterize the molecular components that could contribute to the formation of this barrier. MethodsThree distinct regions in the bovine lens cortex were captured by laser capture microdissection guided by dye penetration. Proteins were digested by endoproteinase Lys C and trypsin. Mass spectrometry-based quantitative proteomic analysis followed by gene ontology (GO) and protein-protein interaction network analysis was performed. ResultsDye penetration showed that lens fiber cells first shrink the extracellular spaces of the broad sides of fiber cells followed by closure of the extracellular space between narrow sides at normalized lens distance (r/a) of 0.9. Accompanying the closure of extracellular space of the broad sides, dramatic proteomic changes were detected including up-regulation of several cell junctional proteins. AQP0 and its interacting partners ERM proteins were among a few proteins that were upregulated accompanying the closure of extracellular space of the narrow sides suggesting a particularly important role for the major lens membrane protein AQP0 in controlling the narrowing of the extracellular spaces between lens fiber cells. The results also provided important information related to biological processes that occur during fiber cell differentiation such as organelle degradation, cytoskeletal remodeling and GSH synthesis. ConclusionsThe formation of lens extracellular diffusion barrier is accompanied by significant membrane and cytoskeletal protein remodeling.

biochemistry↗

bioRxiv2021

Functional cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein

Hendra virus (HeV) and Nipah virus (NiV), the prototypic members of the Henipavirus (HNV) genus, are emerging, zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans (Eaton et al., 2006). While several research groups have made strides in developing candidate vaccines and therapeutics against henipaviruses, such countermeasures have not been licensed for human use, and significant gaps in knowledge about the human immune response to these viruses exist. To address these gaps, we isolated a large panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior occupation-related exposure to the equine HeV vaccine (Equivac(R) HeV). Competition-binding and hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies identified at least six distinct antigenic sites on the HeV/NiV receptor binding protein (RBP) that are recognized by human mAbs. Antibodies recognizing multiple antigenic sites potently neutralized NiV and/or HeV isolates in vitro. The most potent class of cross-reactive antibodies achieved neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3. Antibodies from this class mimic receptor binding by inducing a receptor-bound conformation to the HeV-RBP protein tetramer, exposing an epitope that appears to lie hidden in the interface between protomers within the HeV-RBP tetramer. Antibodies that recognize this cryptic epitope potently neutralized HeV and NiV. Flow cytometric studies using cell-surface-displayed HeV-RBP protein showed that cross-reactive, neutralizing mAbs from each of these classes cooperate for binding. In a highly stringent hamster model of NiVB infection, antibodies from both classes reduced morbidity and mortality and achieved synergistic protection in combination and provided therapeutic benefit when combined into two bispecific platforms. These studies identified multiple candidate mAbs that might be suitable for use in a cocktail therapeutic approach to achieve synergistic antiviral potency and reduce the risk of virus escape during treatment.

immunology↗

bioRxiv2020

Changes to zonular tension alters the subcellular distribution of AQP5 in regions of influx and efflux of water in the rat lens.

PurposeThe lens utilizes circulating fluxes of ions and water that enter the lens at both poles and exit at the equator to maintain its optical properties. We have mapped the subcellular distribution of the lens aquaporins (AQP0, 1, & 5) in these water influx and efflux zones and investigated how their membrane location is affected by changes in tension applied to the lens by the zonules. MethodsImmunohistochemistry using AQP antibodies was performed on axial sections obtained from rat lenses that had been removed from the eye and then fixed, or were fixed in situ to maintain zonular tension. Zonular tension was pharmacologically modulated by applying either tropicamide (increased), or pilocarpine (decreased). AQP labelling was visualized using confocal microscopy. ResultsModulation of zonular tension had no effect on AQP1 or AQP0 labelling in either the water efflux, or influx zones. In contrast, AQP5 labelling changed from membranous to cytoplasmic in response to both mechanical and pharmacologically induced reductions in zonular tension in both the efflux zone, and anterior (but not posterior) influx zone associated with the lens sutures. ConclusionsAltering zonular tension dynamically regulates the membrane trafficking of AQP5 in the efflux and anterior influx zones to potentially change the magnitude of circulating water fluxes in the lens.

physiology↗

bioRxiv2020

Localization of the Lens Intermediate Filament Switch by Imaging Mass Spectrometry

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.

biochemistry↗

bioRxiv2020

Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High Resolution Imaging Mass Spectrometry

The human retina evolved to facilitate complex visual tasks. It supports vision at light levels ranging from starlight to sunlight, and its supporting tissues and vasculature regulate plasma-delivered lipophilic essentials for vision, including retinoids (vitamin A derivatives). The human retina is of particular interest because of its unique anatomic specializations for high-acuity and color vision that are also vulnerable to prevalent blinding diseases. The retinas exquisite cellular architecture is composed of numerous cell types that are aligned horizontally, giving rise to structurally distinct cell, synaptic, and vascular layers that are visible in histology and in diagnostic clinical imaging. Suitable for retinal investigations, MALDI imaging mass spectrometry (IMS) technologies are now capable of providing images at low micrometer spatial resolution with high levels of chemical specificity. In this study, a multimodal imaging approach combined with a recently developed method of high accuracy multi-image registration was used to define the localization of lipids in human retina tissue at laminar, cellular, and sub-cellular levels. Data acquired by IMS combined with autofluorescence and bright-field microscopy of human retina sections in macular and peripheral regions indicate differences in distributions and abundances of lipid species across and within single cell types. Of note is localization of signals within specific layers of macula, localization within different compartments of photoreceptors and RPE, complementarity of signals between macular retina and non-macular RPE, and evidence that lipids differing by a single double bond can have markedly different distributions.

biochemistry↗