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The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or “discs”, located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called “incisures”. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

The photoreceptor outer segment is a modified cilium filled with hundreds of flattened “disc” membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of twenty key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio amongst all twenty proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

The photoreceptor outer segment is a modified cilium filled with hundreds of flattened “disc” membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment.

The small GTPase Arl3 is important for the enrichment of lipidated proteins to primary cilia, including the outer segment of photoreceptors. Human mutations in the small GTPase Arl3 cause both autosomal recessive and dominant inherited retinal dystrophies. We discovered that dominant mutations result in increased active G-protein—Arl3-D67V has constitutive activity and Arl3-Y90C is fast cycling—and their expression in mouse rods resulted in a displaced nuclear phenotype due to an aberrant Arl3-GTP gradient. Using multiple strategies, we go on to show that removing or restoring the Arl3-GTP gradient within the cilium is sufficient to rescue the nuclear migration defect. Together, our results reveal that a Arl3 ciliary gradient is involved in proper positioning of photoreceptor nuclei during retinal development.

Strong evidence suggests that dysregulated lipid metabolism involving dysfunction of the retinal pigmented epithelium (RPE) underlies the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the elderly. A hallmark of AMD is the overproduction of lipid- and protein-rich extracellular deposits that accumulate in the extracellular matrix (Bruch's membrane (BrM)) adjacent to the RPE. We analyzed apolipoprotein A-1 (ApoA-1)-containing lipoproteins isolated from BrM of elderly human donor eyes and found a unique proteome, distinct from high-density lipoprotein (HDL) isolated from donor plasma of the same individuals. The most striking difference is higher concentrations of ApoB and ApoE, which bind to glycosaminoglycans. We hypothesize that this interaction promotes lipoprotein deposition onto BrM glycosaminoglycans, initiating downstream effects that contribute to RPE dysfunction/death. We tested this hypothesis using two potential therapeutic strategies to alter the lipoprotein/protein profile of these extracellular deposits. First, we used short heparan sulfate oligosaccharides to remove lipoproteins already deposited in both the extracellular matrix of RPE cells and aged donor BrM tissue. Second, an ApoA-1 mimetic, 5A peptide, was demonstrated to modulate the composition and concentration of apolipoproteins secreted from primary porcine RPE cells. Significantly, in a mouse model of AMD, this 5A peptide altered the proteomic profile of circulating HDL and ameliorated some of the potentially harmful changes to the protein composition resulting from the high-fat, high-cholesterol diet in this model. Together, these results suggest that targeting HDL interactions with BrM represents a new strategy to slow AMD progression in humans.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease., Here the authors present an approach that can reveal the full complement of mRNA isoforms encoded by individual genes, and they identify a major isoform of the retinal degeneration gene CRB1 which functions at the cell-cell junctions of the outer limiting membrane to promote photoreceptor survival.

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open-angle glaucoma patients compared to non-glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti-apoptotic and anti-TGF-β activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF-β2-induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride-induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF-α-induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF-β induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.

The outer segment (OS) organelle of vertebrate photoreceptors is a highly specialized cilium evolved to capture light and initiate light response. The plasma membrane which envelopes the OS plays vital and diverse roles in supporting photoreceptor function and health. However, little is known about the identity of its protein constituents, as this membrane cannot be purified to homogeneity. In this study, we used the technique of protein correlation profiling to identify unique OS plasma membrane proteins. To achieve this, we used label-free quantitative MS to compare relative protein abundances in an enriched preparation of the OS plasma membrane with a preparation of total OS membranes. We have found that only five proteins were enriched at the same level as previously validated OS plasma membrane markers. Two of these proteins, TMEM67 and TMEM237, had not been previously assigned to this membrane, and one, embigin, had not been identified in photoreceptors. We further showed that embigin associates with monocarboxylate transporter MCT1 in the OS plasma membrane, facilitating lactate transport through this cellular compartment., Graphical Abstract, Highlights • The unique proteome of the photoreceptor outer segment plasma membrane is identified. • TMEM67, TMEM237, and embigin are novel unique components of this membrane. • Embigin in this membrane is associated with the monocarboxylate transporter MCT1. • The photoreceptor outer segment likely facilitates lactate transport., In Brief The plasma membrane which envelopes the light-sensitive outer segment organelle of vertebrate photoreceptor cells plays diverse roles in supporting photoreceptor function and health. Protein correlation profiling of this membrane revealed a surprisingly small number of unique protein components. Among them are TMEM67 and TMEM237, whose mutations are associated with various syndromic ciliopathies, and embigin found to be associated with the monocarboxylate transporter MCT1. The MCT1–embigin complex likely facilitates lactate transport through this cellular compartment.

The molecular and cellular basis for cataract development in mice lacking dystrophin, a scaffolding protein that links the cytoskeleton to the extracellular matrix, is poorly understood. In this study, we characterized lenses derived from the dystrophin-deficient mdx(3cv) mouse model. Expression of Dp71, a predominant isoform of dystrophin in the lens, was induced during lens fiber cell differentiation. Dp71 was found to co-distribute with dystroglycan, connexin-50 and 46, aquaporin-0, and NrCAM as a large cluster at the center of long arms of the hexagonal fibers. Although mdx(3cv) mouse lenses exhibited dramatically reduced levels of Dp71, only older lenses revealed punctate nuclear opacities compared to littermate wild type (WT) lenses. The levels of dystroglycan, syntrophin, and dystrobrevin which comprise the dystrophin-associated protein complex (DAPC), and NrCAM, connexin-50, and aquaporin-0, were significantly lower in the lens membrane fraction of adult mdx(3cv) mice compared to WT mice. Additionally, decreases were observed in myosin light chain phosphorylation and lens stiffness together with a significant elevation in the levels of utrophin, a functional homolog of dystrophin in mdx(3cv) mouse lenses compared to WT lenses. The levels of perlecan and laminin (ligands of α-dystroglycan) remained normal in dystrophin-deficient lens fibers. Taken together, although mdx(3cv) mouse lenses exhibit only minor defects in lens clarity possibly due to a compensatory increase in utrophin, the noted disruptions of DAPC, stability, and organization of membrane integral proteins of fibers, and stiffness of mdx(3cv) lenses reveal the importance of dystrophin and DAPC in maintaining lens clarity and function.

Individuals with pseudoexfoliation (PEX) syndrome exhibit various connective tissue pathologies associated with dysregulated extracellular matrix homeostasis. PEX glaucoma is a common, aggressive form of open-angle glaucoma resulting from the deposition of fibrillary material in the conventional outflow pathway. However, the molecular mechanisms that drive pathogenesis and genetic risk remain poorly understood. PEX glaucoma-associated single-nucleotide polymorphisms are located in and affect activity of the promoter of LOXL1-AS1, a long non-coding RNA (lncRNA). Nuclear and non-nuclear lncRNAs regulate a host of biological processes, and when dysregulated, contribute to disease. Here we report that LOXL1-AS1 localizes to the nucleus where it selectively binds to the mRNA processing protein, heterogeneous nuclear ribonucleoprotein-L (hnRNPL). Both components of this complex are critical for the regulation of global gene expression in ocular cells, making LOXL1-AS1 a prime target for investigation in PEX syndrome and glaucoma.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier, provides nutrients, recycles visual pigment, and removes spent discs from the photoreceptors, among many other functions. Because of these critical roles in visual homeostasis, the RPE is a principal location of disease-associated changes in age-related macular degeneration (AMD), emphasizing its importance for study in both visual health and disease. Unfortunately, there are no early indicators of AMD or disease progression, a void that could be filled by the development of early AMD biomarkers. Exosomes are lipid bilayer membrane vesicles of nanoscale sizes that are released in a controlled fashion by cells and carry out a number of extra- and intercellular activities. In the RPE they are released from both the apical and basal sides, and each source has a unique signature/content. Exosomes released from the basolateral side of RPE cells enter the systemic circulation via the choroid and thus represent a potential source of retinal disease biomarkers in blood. Here we discuss the potential of targeted immunocapture of eye-derived exosomes and other small extracellular vesicles from blood for eye disease biomarker discovery.

The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a “lamellipodium-like” mechanism.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers.

Glaucoma, a leading cause of irreversible blindness, is commonly associated with elevated intraocular pressure (IOP) due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although dysregulated production and organization of extracellular matrix (ECM) is presumed to increase resistance to AH outflow and elevate IOP by altering TM cell contractile and adhesive properties, it is not known whether regulation of ECM protein phosphorylation via the secretory vertebrate lonesome kinase (VLK) influences TM cellular characteristics. Here, we tested this possibility. Experiments carried out in this study reveal that the 32 kDa protein is a prominent VLK isoform detectable in lysates and conditioned media (CM) of human TM cells. Increased levels of VLK were observed in CM of TM cells subjected to cyclic mechanical stretch, or treated with dexamethasone, TGF-β2, and TM cells expressing constitutively active RhoA GTPase. Downregulation of VLK expression in TM cells using siRNA decreased tyrosine phosphorylation (TyrP) of ECM proteins and focal adhesions, and induced changes in cell shape in association with reduced levels of actin stress fibers and phospho-paxillin. VLK was also demonstrated to regulate TGF-β2-induced TyrP of ECM proteins. Taken together, these results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins, and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers, and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma. J. Cell. Physiol. 232: 2447–2460, 2017. © 2016 Wiley Periodicals, Inc.

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier, provides nutrients, recycles visual pigment, and removes spent discs from the photoreceptors, among many other functions. Because of these critical roles in visual homeostasis, the RPE is a principal location of disease-associated changes in age-related macular degeneration (AMD), emphasizing its importance for study in both visual health and disease. Unfortunately, there are no early indicators of AMD or disease progression, a void that could be filled by the development of early AMD biomarkers. Exosomes are lipid bilayer membrane vesicles of nanoscale sizes that are released in a controlled fashion by cells and carry out a number of extra- and intercellular activities. In the RPE they are released from both the apical and basal sides, and each source has a unique signature/content. Exosomes released from the basolateral side of RPE cells enter the systemic circulation via the choroid and thus represent a potential source of retinal disease biomarkers in blood. Here we discuss the potential of targeted immunocapture of eye-derived exosomes and other small extracellular vesicles from blood for eye disease biomarker discovery.

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The molecular and cellular basis for cataract development in mice lacking dystrophin, a scaffolding protein that links the cytoskeleton to the extracellular matrix, is poorly understood. In this study, we characterized lenses derived from the dystrophin-deficient mdx3cv mouse model. Expression of Dp71, a predominant isoform of dystrophin in the lens, was induced during lens fiber cell differentiation. Dp71 was found to co-distribute with dystroglycan, connexin-50 and 46, aquaporin-0, and NrCAM as a large cluster at the center of long arms of the hexagonal fibers. Although mdx3cv mouse lenses exhibited dramatically reduced levels of Dp71, only older lenses revealed punctate nuclear opacities compared to littermate wild type (WT) lenses. The levels of dystroglycan, syntrophin, and dystrobrevin which comprise the dystrophin-associated protein complex (DAPC), and NrCAM, connexin-50, and aquaporin-0, were significantly lower in the lens membrane fraction of adult mdx3cv mice compared to WT mice. Additionally, decreases were observed in myosin light chain phosphorylation and lens stiffness together with a significant elevation in the levels of utrophin, a functional homolog of dystrophin in mdx3cv mouse lenses compared to WT lenses. The levels of perlecan and laminin (ligands of α-dystroglycan) remained normal in dystrophin-deficient lens fibers. Taken together, although mdx3cv mouse lenses exhibit only minor defects in lens clarity possibly due to a compensatory increase in utrophin, the noted disruptions of DAPC, stability, and organization of membrane integral proteins of fibers, and stiffness of mdx3cv lenses reveal the importance of dystrophin and DAPC in maintaining lens clarity and function.

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin–proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness., Proteasomal overload can be found in a broad spectrum of mouse models of retinal degeneration. Here the authors find that overexpressing the PA28α subunit of the 11S proteasome cap increased the number of surviving functional photoreceptor cells in a mouse model of retinal degeneration bearing the P23H mutation in rhodopsin.

Purpose Lysophosphatidic acid (LPA), a bioactive lipid, has been shown to increase resistance to aqueous humor outflow (AH) through the trabecular meshwork (TM). The molecular basis for this response of the TM to LPA, however, is not completely understood. In this study, we explored the possible involvement of mechanosensitive Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding domain (TAZ), transcriptional activation in extracellular matrix (ECM) production by LPA-induced contractile activity in human TM cells (HTM). Methods The responsiveness of genes encoding LPA receptors (LPARs), LPA hydrolyzing lipid phosphate phosphatases (LPPs), and the LPA-generating autotaxin (ATX) to cyclic mechanical stretch in HTM cells, was evaluated by RT-quantitative (q)PCR. The effects of LPA and LPA receptor antagonists on actomyosin contractile activity, activation of YAP/TAZ, and levels of connective tissue growth factor (CTGF), and Cyr61 and ECM proteins in HTM cells were determined by immunoblotting, mass spectrometry, and immunofluorescence analyses. Results Cyclic mechanical stretch significantly increased the expression of several types of LPARs, LPP1, and ATX in HTM cells. LPA and LPA receptor-dependent contractile activity led to increases in both, the protein levels and activation of YAP/TAZ, and increased the levels of CTGF, Cyr61, α-smooth muscle actin (α-SMA), and ECM proteins in HTM cells. Conclusions The results of this study reveal that LPA and its receptors stimulate YAP/TAZ transcriptional activity in HTM cells by modulating cellular contractile tension, and augment expression of CTGF that in turn leads to increased production of ECM. Therefore, YAP/TAZ-induced increases in CTGF and ECM production could be an important molecular mechanism underlying LPA-induced resistance to AH outflow and ocular hypertension.

The heterotrimeric G-protein transducin mediates visual signaling in vertebrate photoreceptor cells. Many aspects of the function of transducin were learned from knock-out mice lacking its individual subunits. Of particular interest is the knockout of its rod-specific γ-subunit (Gγ1). Two studies using independently generated mice documented that this knockout results in a considerable >60-fold reduction in the light sensitivity of affected rods, but provided different interpretations of how the remaining α-subunit (Gαt) mediates phototransduction without its cognate Gβ1γ1-subunit partner. One study found that the light sensitivity reduction matched a corresponding reduction in Gαtcontent in the light-sensing rod outer segments and proposed that Gαtactivation is supported by remaining Gβ1associating with other Gγ subunits naturally expressed in photoreceptors. In contrast, the second study reported the same light sensitivity loss but a much lower, only approximately sixfold, reduction of Gαtand proposed that the light responses of these rods do not require Gβγ at all. To resolve this controversy and elucidate the mechanism driving visual signaling in Gγ1knock-out rods, we analyzed both mouse lines side by side. We first determined that the outer segments of both mice have identical Gαtcontent, which is reduced ∼65-fold from the wild-type (WT) level. We further demonstrated that the remaining Gβ1is present in a complex with endogenous Gγ2and Gγ3subunits and that these complexes exist in wild-type rods as well. Together, these results argue against the idea that Gαtalone supports light responses of Gγ1knock-out rods and suggest that Gβ1γ1is not unique in its ability to mediate vertebrate phototransduction.

The bacterium Lysobacter species strain XL1 is known as a producer of extracellular lytic enzymes, which are capable of degrading cell wall components of other bacteria and simple eukaryotes. This ability determines the ecological, medical and agricultural relevance of Lysobacter sp. XL1. However, the molecular mechanism of secretion of lytic exoenzymes from Lysobacter cells is yet unknown, which in turn necessitates the search of protein–protein interactions that occur during exoenzyme secretion. The current paper is concerned with investigation of protein complexes that are likely formed during the secretion of AlpB lytic protease from the cells of Lysobacter sp. XL1. In this study, we have optimized the method of stabilization of protein complexes formed in the intact cells of Lysobacter sp. XL1 by using crosslinking reagent dithiobis(succinimidylpropionate) (DSP) and detected DSP-linked protein complexes by the monoclonal antibodies against AlpB propeptide.

Visual signal transduction takes place on the surface of flat membrane vesicles called photoreceptor discs, which reside inside the light-sensitive outer segment organelle of vertebrate photoreceptor cells. Although biochemical studies have indicated that discs are built with a handful of highly specialized proteins, proteomic studies have yielded databases consisting of hundreds of entries. We addressed this controversy by employing protein correlation profiling, which allows identification of unique components of organelles that can be fractionated but not purified to absolute homogeneity. We subjected discs to sequential steps of fractionation and identified the relative amounts of proteins in each fraction by label-free quantitative mass spectrometry. This analysis demonstrated that the photoreceptor disc proteome contains only eleven components, which satisfy the hallmark criterion for being unique disc-resident components: the retention of a constant molar ratio among themselves across fractionation steps. Remarkably, one of them is PRCD, a protein whose mutations have been shown to cause blindness, yet cellular localization remained completely unknown. Identification of PRCD as a novel disc-specific protein facilitates understanding its functional role and the pathobiological significance of its mutations. Our study provides a striking example how protein correlation profiling allows a distinction between constitutive components of cellular organelles and their inevitable contaminants.

Transparency of the ocular lens depends on symmetric packing and membrane organization of highly elongated hexagonal fiber cells. These cells possess an extensive, well-ordered cortical cytoskeleton to maintain cell shape and to anchor membrane components. Periaxin (Prx), a PDZ domain protein involved in myelin sheath stabilization, is also a component of adhaerens plaques in lens fiber cells. Here we show that Prx is expressed in lens fibers and exhibits maturation dependent redistribution, clustering discretely at the tricellular junctions in mature fiber cells. Prx exists in a macromolecular complex with proteins involved in membrane organization including ankyrin-B, spectrin, NrCAM, filensin, ezrin and desmoyokin. Importantly, Prx knockout mouse lenses were found to be softer and more easily deformed than normal lenses, revealing disruptions in fiber cell hexagonal packing, membrane skeleton and membrane stability. These observations suggest a key role for Prx in maturation, packing, and membrane organization of lens fiber cells. Hence, there may be functional parallels between the roles of Prx in membrane stabilization of the myelin sheath and the lens fiber cell.

The remarkable ability of our vision to function under ever-changing conditions of ambient illumination is mediated by multiple molecular mechanisms regulating the light sensitivity of rods and cones. One such mechanism involves massive translocation of signaling proteins, including the G-protein transducin, into and out of the light-sensitive photoreceptor outer segment compartment. Transducin translocation extends the operating range of rods, but in cones transducin never translocates, which is puzzling because cones typically function in much brighter light than rods. Using genetically manipulated mice in which the rates of transducin activation and inactivation were altered, we demonstrate that, like in rods, transducin translocation in cones can be triggered when transducin activation exceeds a critical level, essentially saturating the photoresponse. However, this level is never achieved in wild-type cones: their superior ability to tightly control the rates of transducin activation and inactivation, responsible for avoiding saturation by light, also accounts for the prevention of transducin translocation at any light intensity.

The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its gamma-subunit (Pgamma), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of Pgamma are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (Pgamma78-87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the Pgamma C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of Pgamma inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact alpha2 helix of Pgamma stabilize binding of C-terminal Pgamma peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the Pgamma molecule and tested for activation by transducin, we found that the C-terminal region (Pgamma63-87) by itself could not be displaced but that transducin could relieve inhibition of certain Pgamma truncation mutants. Our results are consistent with two distinct mechanisms of Pgamma inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of Pgamma to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of Pgamma to effectively relieve its inhibitory constraint.

Variations in the complement factor H (CFH) gene are tightly associated with age-related macular degeneration (AMD) across diverse populations. Of the many nonsynonymous coding variants in CFH, two are most strongly associated with increased risk of AMD: isoleucine 62 to valine (I62V) and tyrosine 402 to histidine (Y402H). Detection of these variations in a patient's blood is important for a risk assessment of AMD and disease prognosis. However, traditional methods of genetic analysis cannot be used for measuring CFH allotypes in some sources of human plasma and other biological fluids not containing DNA. The purpose was to develop a protein-based method of detecting CFH allotypes.A combination of a single-step affinity enrichment of CFH, gel separation, and mass spectrometry identification of the CFH peptides spanning amino acids at positions 62 and 402 was used to identify individual CFH allotypes.The CFH isoforms V62, I62, H402, and Y402 were reliably detected based on identification of tryptic peptides with masses of 1148.59 Da, 1162.60 Da, 2031.88 Da, and 2057.88 Da, respectively, using MALDI-TOF-TOF. The presence or absence pattern of these peptides in mass spectra of different CFH samples robustly correlated with all nine genotypes of CFH, as a result of variations at positions 62 and 402.A rapid and sensitive method has been developed for detection of V62, I62, H402, and Y402 variants of CFH in human plasma samples using mass spectrometry. This method can be used in clinical laboratories equipped with a basic inexpensive mass spectrometer capable of performing peptide fingerprinting.

To understand lens fiber cell elongation- and differentiation-associated cytoskeletal remodeling, here we identified and characterized the major protein components of lens fiber cell Triton X-100 insoluble fraction by mass spectrometry and immunoblot analysis. This analysis identified spectrin, filensin, vimentin, tubulin, phakinin, and beta-actin as major cytoskeletal proteins in the lens fibers. Importantly, ezrin, radixin, and moesin (ERM), heat-shock cognate protein 70, and beta/gamma-crystallins were identified as major cytoskeletal-associated proteins. ERM proteins were confirmed to exist as active phosphorylated forms that exhibited intense distribution in the organelle free-zone fibers. Furthermore, ERM protein phosphorylation was found to be dramatically reduced in Rho GTPase-targeted transgenic mouse lenses. These data identify the ERM proteins, which cross-link the plasma membrane and actin, as major and stable cytoskeletal-associated proteins in lens fibers, and indicate a potential role(s) for the ERMs in fiber cell actin cytoskeletal and membrane organization.

SummaryThe exosome complex plays a central and essential role in RNA metabolism. However, comprehensive studies of exosome substrates and functional analyses of its subunits are lacking. Here, we demonstrate that as opposed to yeast and metazoans the plant exosome core possesses an unanticipated functional plasticity and present a genome-wide atlas of Arabidopsis exosome targets. Additionally, our study provides evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants, reveals unexpected aspects of stable structural RNA metabolism, and uncovers numerous novel exosome substrates. These include a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described and belong to a layer of the transcriptome that can only be visualized upon inhibition of exosome activity. These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes.

Light causes massive translocation of G-protein transducin from the light-sensitive outer segment compartment of the rod photoreceptor cell. Remarkably, significant translocation is observed only when the light intensity exceeds a critical threshold level. We addressed the nature of this threshold using a series of mutant mice and found that the threshold can be shifted to either a lower or higher light intensity, dependent on whether the ability of the GTPase-activating complex to inactivate GTP-bound transducin is decreased or increased. We also demonstrated that the threshold is not dependent on cellular signaling downstream from transducin. Finally, we showed that the extent of transducin α subunit translocation is affected by the hydrophobicity of its acyl modification. This implies that interactions with membranes impose a limitation on transducin translocation. Our data suggest that transducin translocation is triggered when the cell exhausts its capacity to activate transducin GTPase, and a portion of transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. Overall, the threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less-sensitive energy-saving mode beneficial in bright light, when vision is dominated by cones.

The R7 subfamily of the regulators of G protein signaling (RGS) proteins is represented by four members broadly expressed in the mammalian nervous system. Here we report that in the brain all four R7 proteins form tight complexes with a previously unidentified protein, which we call the R7-binding protein or R7BP. We initially identified R7BP as a protein co-precipitating with the R7 protein, RGS9, from extracts obtained from the striatal region of the brain. We further showed that R7BP forms a tight complex with RGS9 in vitro and that this binding occurs via the N-terminal DEP domain of RGS9. R7BP is expressed throughout the entire central nervous system but not in any of the tested non-neuronal tissues. All four R7 RGS proteins co-precipitate with R7BP from brain extracts and recombinant R7 proteins bind recombinant R7BP with high efficiency. The closest homolog of R7BP is R9AP which was previously found to interact with RGS9 in photoreceptors. Both R7BP and R9AP are related to the syntaxin subfamily of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins involved in vesicular trafficking and exocytosis. In photoreceptors R9AP regulates several critical properties of RGS9 including its intracellular targeting, stability and catalytic activity. This suggests that R7BP interactions with R7 proteins in the brain may also bear major functional significance.

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.

Purpose. Complement activation is implicated in the pathogenesis of age-related macular degeneration (AMD). Apolipoprotein E (ApoE) and complement activation products such as membrane attack complex (MAC) are present in eyes of individuals with AMD. Herein, we investigated the effect of complement activation on induction of ApoE accumulation in human retinal pigment epithelial (RPE) cells. Methods. Cultured human RPE cells were primed with a complement-fixing antibody followed by treatment with C1q-depleted (C1q-Dep) human serum to elicit alternative pathway complement activation. Controls included anti-C5 antibody-treated serum and heat-inactivated C1q-Dep. Total protein was determined on RPE cell extracts, conditioned media, and extracellular matrix (ECM) by Western blot. ApoE and MAC colocalization was assessed on cultured RPE cells and human eyes by immunofluorescent stain. ApoE mRNA expression was evaluated by quantitative PCR (qPCR). Results. Complement challenge upregulated cell-associated ApoE, but not apolipoprotein A1. ApoE accumulation was blocked by anti-C5 antibody and enhanced by repetitive complement challenge. ApoE mRNA levels were not affected by complement challenge. ApoE was frequently colocalized with MAC in complement-treated cells and drusen from human eyes. ApoE was released into complement-treated conditioned media after a single complement challenge and accumulated on ECM after repetitive complement challenge. Conclusions. Complement challenge induces time-dependent ApoE accumulation in RPE cells. An understanding of the mechanisms by which complement affects RPE ApoE accumulation may help to better explain drusen composition, and provide insights into potential therapeutic targets.

RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein alpha subunits. The complex of RGS9 with type 5 G protein beta subunit (G beta 5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-G beta 5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-G beta 5 make opposite contributions toward this selectivity. G beta 5 bound to the G protein gamma subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action.

Purpose To determine the role and regulation of growth differentiation factor-15 (GDF-15), a TGF-β-related cytokine in human trabecular meshwork (TM) cells in the context of aqueous humor (AH) outflow and IOP. Methods Regulation of expression by external cues, and the distribution and secretion of GDF-15 by human TM primary cell cultures, and the effects of recombinant (r) GDF-15 on TM cell contractile characteristics, actin cytoskeleton, cell adhesion, extracellular matrix (ECM), α-smooth muscle actin (αSMA), SMAD signaling, and gene expression were determined by immunoblot, immunofluorescence, mass spectrometry, cDNA microarray, and real-time quantitative PCR (RT-qPCR) analyses. Results Growth differentiation factor-15, a common constituent of ECM derived from the human TM cells, was confirmed to be distributed throughout the conventional aqueous humor outflow pathway of the human eye. Growth differentiation factor-15 protein levels were significantly increased in human TM cells in response to TGF-β2, dexamethasone, endothelin-1, lysophosphatidic acid, TNF-α, IL-1β treatment, and by cyclic mechanical stretch. Stimulation of human TM cells with rGDF-15 caused a significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, SMAD signaling, gene expression, and the levels of αSMA and ECM proteins. Conclusions The results of this study, including a robust induction of GDF-15 expression by several external factors known to elevate IOP, and rGDF-15-induced increase in contractility, cell adhesion, and the levels of ECM proteins and αSMA in TM cells, collectively suggest a potential role for GDF-15 in homeostasis and dysregulation of AH outflow and IOP in normal and glaucomatous eyes, respectively.

To understand the dynamics of conformational changes during G protein activation, surface exposed cysteine residues on Galpha were fluorescently labeled. Limited trypsinolysis and mutational analysis of recombinant Galphat/Galphai1 determined that two cysteines are the major fluorescent labeling sites, Cys210, located in the switch II region, and Cys347 at the C terminus. Mutants with serines replacing Cys210 (Chi6a) and Cys347 (Chi6b) were single fluorescently labeled with lucifer yellow (LY), while a double mutant (Chi6ab) was no longer labeled. When Chi6b was labeled with LY on Cys210, AlF4- caused a 220% increase in LY fluorescence, indicating that the fluorescent group at Cys210 is a reporter of conformational change in the switch II region. Chi6a labeled at Cys347 also showed an AlF4--dependent increase in LY fluorescence (91%), indicating that Galpha activation leads to a conformational change at the COOH terminus. Preactivation of the protein with AlF4- before labeling led to a decreased incorporation of LY into Cys347 suggesting that Galpha activation buries Cys347. This COOH-terminal conformational change may provide the structural basis for communication between the GDP-binding site on Galpha and activated receptors, and may contribute to dissociation of activated Galpha subunit from activated receptor.

The crystal structure of the catalytic domain of adenylyl cyclase in complex with its activator Gsα suggests a surprising allosteric mechanism of activation by formation of a new catalytic site at the interface between conserved dimers of the soluble adenylyl cyclase domains.

The recognition between G protein and cognate receptor plays a key role in specific cellular responses to environmental stimuli. Here we explore specificity in receptor-G protein coupling by taking advantage of the ability of the 5-hydroxytryptamine1B (5-HT1B) receptor to discriminate between G protein heterotrimers containing Galphai1 or Galphat. Gi1 can interact with the 5-HT1B receptor and stabilize a high affinity agonist binding state of this receptor, but Gt cannot. A series of Galphat/Galphai1 chimeric proteins have been generated in Escherichia coli, and their functional integrity has been reported previously (Skiba, N. P., Bae, H., and Hamm, H. E. (1996) J. Biol. Chem. 271, 413-424). We have tested the functional coupling abilities of the Galphat/Galphai1 chimeras to 5-HT1B receptors using high affinity agonist binding and receptor-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. In the presence of betagamma subunits, amino acid residues 299-318 of Galphai1 increase agonist binding to the 5-HT1B receptor and receptor stimulation of GTPgammaS binding. Moreover, Galphai1 containing only Galphat amino acid sequences from this region does not show any coupling ability to 5-HT1B receptors. Our studies suggest that the alpha4 helix and alpha4-beta6 loop region of Galphas are an important region for specific recognition between receptors and Gi family members.

Inherited retinal degenerations, caused by mutations in over 100 individual genes, affect approximately 2 million people worldwide. Many of the underlying mutations cause protein misfolding or mistargeting in affected photoreceptors. This places an increased burden on the protein folding and degradation machinery, which may trigger cell death. We analyzed how these cellular functions are affected in degenerating rods of the transducin γ-subunit (Gγ 1 ) knockout mouse. These rods produce large amounts of transducin β-subunit (Gβ 1 ), which cannot fold without Gγ 1 and undergoes intracellular proteolysis instead of forming a transducin βγ-subunit complex. Our data revealed that the most critical pathobiological factor leading to photoreceptor cell death in these animals is insufficient capacity of proteasomes to process abnormally large amounts of misfolded protein. A decrease in the Gβ 1 production in Gγ 1 knockout rods resulted in a significant reduction in proteasomal overload and caused a striking reversal of photoreceptor degeneration. We further demonstrated that a similar proteasomal overload takes place in photoreceptors of other mutant mice where retinal degeneration has been ascribed to protein mistargeting or misfolding, but not in mice whose photoreceptor degenerate as a result of abnormal phototransduction. These results establish the prominence of proteasomal insufficiency across multiple degenerative diseases of the retina, thereby positioning proteasomes as a promising therapeutic target for treating these debilitating conditions.

The G protein transducin has been an often-used model for biochemical, structural, and mechanistic studies of G protein function. Experimental studies have been limited, however, by the inability to express quantities of mutants in heterologous systems with ease. In this study we have made a series of G alpha t/G alpha i1 chimeras differing at as few as 11 positions from native G alpha t. Ten chimeras are properly folded, contain GDP, can assume an A1F4(-)-induced activated conformation, and interact with beta gamma t and light-activated rhodopsin. They differ dramatically in their affinity for GDP, from Gi-like (initial rates 225 mumol/mol s) to Gt-like (initial rates 4.9 mumol/mol s). We have used these chimeras to define contact sites on G alpha t with the effector enzyme cGMP phosphodiesterase. G alpha t GTP but not G alpha t GDP activates it by removing the phosphodiesterase (PDE) gamma inhibitory subunit. In solution, G alpha t GTP interacts with PDE gamma (Kd 12 nM), while G alpha t GDP binds PDE gamma more weakly (Kd 0.88 microM). The interaction of G alpha i GDP with PDE gamma is undetectable, but G alpha i GDP-A1F4- interacts weakly with PDE gamma (Kd 2.4 microM). Using defined G alpha t/G alpha i chimeras, we have individuated the regions on G alpha t most important for interaction with PDE gamma in the basal and activated states. The G alpha t sequence encompassing alpha helix 3 and the alpha 3/beta 5 loop contributes most binding energy to interaction with PDE gamma. Another composite P gamma interaction site is the conserved switch, through which the GTP-bound G alpha t as well as G alpha i1 interact with P gamma. Competition studies between PDE gamma and truncated regions of PDE gamma provide evidence for the point-to-point interactions between the two proteins. The amino-terminal 1-45 segment containing the central polycationic region binds to G alpha t's alpha 3 helix and alpha 3/beta 5 loop, while the COOH-terminal region of P gamma, 63-87, binds in concert to the conserved switch regions. The first interaction provides specific interaction with both the GDP- and GTP-liganded G alpha t, while the second one is conserved between G alpha t and G alpha i1 and dependent on the activated conformation.

The interaction between the GTP-bound form of the transducin alpha-subunit (G alpha t) and the gamma-subunit (P gamma) of cGMP phosphodiesterase (PDE) is a key event in effector activation during photon signal transduction. The carboxyl-terminal half of P gamma is involved in interaction with G alpha t as well as in inhibition of PDE activity. Here we have utilized a combination of synthetic peptide and mutagenesis approaches to localize specific regions of the carboxyl-terminal region of P gamma interacting with G alpha t and P alpha beta and have determined residues involved in inhibition of PDE activity. We found that synthetic peptide corresponding to residues 68-87 of P gamma completely inhibit trypsin-activated PDE. The peptide P gamma-63-87 bound to G alpha t GTP gamma S with a Kd of 2.5 microM, whereas the binding of P gamma-68-87 to G alpha tGTP gamma S was approximately 15-fold less (Kd = 40 microM) suggesting that carboxyl-terminal P gamma region 68-87 contains a site for interaction with P alpha beta and also a part of the alpha t binding site. To map G alpha t and P alpha beta sites more precisely within the carboxyl-terminal region, a set of carboxyl-terminal mutants was generated by site-directed mutagenesis. Deletion of residues 63-69 and 70-76 diminished the binding of mutants to alpha t while binding to carboxyl-terminally truncated mutants lacking up to 11 amino acid residues was unchanged. In contrast, carboxyl-terminal truncations of P gamma from delta 1 to delta 11 resulted in a gradual decrease of its inhibitory activity. Thus, the extreme carboxyl-terminal hydrophobic sequence -Ile86-Ile87 together with 9 adjacent residues provides inhibitory interaction of P gamma with P alpha beta. The carboxyl-terminal G alpha tGTP gamma S binding site of P gamma is different from but adjacent to its PDE inhibitory site. During the visual transduction process, G alpha tGTP likely binds to this region of P gamma inducing a displacement of the extreme carboxyl terminus from the inhibitory site on P alpha beta, leading to PDE activation.