two-peeling-methods-for-isolation-photoreceptor-cell-compartments

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

University of Southern California

The intrinsically unstructured protein alpha-synuclein (aS) is prone to misfold into cytotoxic beta-sheet-rich oligomers and amyloid fibrils that underlie the pathogenesis of Lewy body diseases such as Parkinson's disease. An important, recognized fibrillogenesis parameter is amino acid content, whereas the influence of amino acid sequence distribution is not as well understood. The fibril core of aS encompasses five regions of high beta-sheet propensity, termed beta1-beta5. Using four aS variants with identical amino acid compositions but rearranged pseudorepeat motifs, we show that beta2-beta5 sequence clustering, but not order, is important for efficient fibrillogenesis. For molecular species progressing toward the fibrillar state, order invariably increases; i.e., the spatial arrangement of sequence elements becomes restricted. By introducing disulfide bonds in a fibril structure-based manner, we demonstrated that a successful protofibril-to-fibril conversion is dependent upon the spatial arrangement of sequence elements of high beta-sheet propensity. Moreover, a disulfide-linked aS dimer is shown to fibrillize rapidly. We propose that a conformational search underlies the emergence of a fibrillar aS nucleus that is directed by gaps in sequence between beta-sheet regions and the accessible range of spatial beta-sheet arrangements in soluble, prefibrillar oligomers. On the basis of the universal cross-beta-sheet structure of amyloid fibrils, these principles are expected to apply to a wide range of amyloidogenic proteins.

The clustering and spatial arrangement of beta-sheet sequence, but not order, govern alpha-synuclein fibrillogenesis.

The misfolding of the protein α-synuclein (αS) has been implicated in the molecular chain of events leading to Parkinson disease. Physiologically, αS undergoes a transition from a random coil to helical conformation upon encountering synaptic vesicle membranes. On analogous small unilamellar vesicles (SUVs), the conformation of αS is dominated by a single elongated αS helix. However, alternative broken helix states have been postulated, mandating experimental clarification. Here, the upper limit for the free energy difference between elongated and broken helix conformations on SUVs resembling synaptic vesicles was determined to be 1.2 ± 0.4 kcal/mol, which amounts to a population ratio of 7.6:1 between both states (12% broken helices). In response to helix breaks at different positions, αS rearranged in an opportunistic manner, thereby minimizing helix abrogations to as little as one to two turns. Enthalpy and entropy measurements of gel state SUV-αS interactions indicated that broken helix states retain the ability to relieve membrane-packing stress. Thus, broken helix states are a distinct physiological feature of the vesicle-bound αS state, making it a "checkered" protein of multiple parallel conformations. A continuous interconversion between structural states may contribute to pathological αS misfolding.

Alpha-synuclein populates both elongated and broken helix states on small unilamellar vesicles.

Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp(161), Trp(45), and Trp(25)) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (R(H)) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg · ml(-1). We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp(161)) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp(25) and Trp(45) is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.

Dissecting amelogenin protein nanospheres: characterization of metastable oligomers.

Caveolin-1 has an atypical membrane-spanning domain comprising of 34 residues. Caveolin-1 targets to lipid droplets under certain conditions, where they are involved in signaling and cholesterol balance. In the present study, membrane association of synthetic peptides corresponding to the membrane-spanning domain of caveolin-1 has been investigated to obtain an insight into the topology of transmembrane region in the lipid bilayer and the effect of truncations in this sequence, as observed in the targeting to lipid droplets, by using model membranes. Fluorescence studies revealed strong association of the peptide corresponding to the membrane-spanning domain of caveolin-1 with anionic lipids as compared with zwitterionic lipids, which is consistent with the location of this domain in the cytoplasmic side of the plasma membrane. Association of a short 9 residue peptide corresponding to the C-terminus of caveolin-1 membrane-spanning domain with lipid vesicles revealed the importance of this region for association with model membranes. Our investigations indicate that the peptide corresponding to the membrane-spanning domain of caveolin-1 does not span the lipid bilayer. We propose that both caveolin scaffolding domain and transmembrane segment of caveolin-1 contribute to the strong association with the plasma membrane rendering the protein highly detergent resistant.

Interaction of peptides spanning the transmembrane domain of caveolin-1 with model membranes.

Calcite crystals were grown in the presence of full-length amelogenin and during its proteolysis by recombinant human matrix metalloproteinase 20 (rhMMP-20). Recombinant porcine amelogenin (rP172) altered the shape of calcite crystals by inhibiting the growth of steps on the {104} faces and became occluded inside the crystals. Upon co-addition of rhMMP-20, the majority of the protein was digested resulting in a truncated amelogenin lacking the C-terminal segment. In rP172-rhMMP-20 samples, the occlusion of amelogenin into the calcite crystals was drastically decreased. Truncated amelogenin (rP147) and the 25-residue C-terminal domain produced crystals with regular shape and less occluded organic material. Removal of the C-terminal diminished the affinity of amelogenin to the crystals and therefore prevented occlusion. We hypothesize that HAP and calcite interact with amelogenin in a similar manner. In the case of each material, full-length amelogenin binds most strongly, truncated amelogenin binds weakly and the C-terminus alone has the weakest interaction. Regarding enamel crystal growth, the prevention of occlusion into maturing enamel crystals might be a major benefit resulting from the selective cleavage of amelogenin at the C-terminus by MMP-20. Our data have important implications for understanding the hypomineralized enamel phenotype in cases of amelogenesis imperfecta resulting from MMP-20 mutations and will contribute to the design of enamel inspired biomaterials.

Amelogenin processing by MMP-20 prevents protein occlusion inside calcite crystals.

Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence, and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172-SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N-terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS-Rosetta modeling confirm that the highly conserved N-terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 525-535, 2014.

Structural adaptation of tooth enamel protein amelogenin in the presence of SDS micelles.

A random coil-helix transition underlies the association of the presynaptic protein α-synuclein (αS) with curved vesicle membranes to fold Asp2-Ala89 into a continuous helix. To clarify this transition, we examined αS folding cooperativity, helix nucleation and propagation in relation to membrane stabilization and leakage on diverse small unilamellar vesicles. The sequences centering on Phe4 and Tyr39 initiate lipid interactions and the Phe4 region nucleates the helix irrespective of the order of Ser9-Ala89. However, helix propagation is not the sum of individual αS-membrane interactions; it requires non-uniform but balanced sequence distributions of lipid affinities and helix flexibility. The attained helix propagation, like folding cooperativity, depends distinctly on membrane lipid composition and correlates to the degree of αS-conferred membrane stabilization. Contrary to classical coil-helix folding thermodynamics, helix propagation proceeds with a small gain in free energy relative to helix nucleation indicating that its binding enthalpy is expended to compensate a high entropic cost of reducing lipid-packing defects in the curved membrane. Non-saturating lipid conditions or rigidification of the αS helix triggers an increase in small unilamellar vesicle membrane leakage. Thus, αS folding parameters appear highly optimized and closely matched to stabilize and protect its target membrane. Aging-associated changes in lipid and αS concentrations may therefore alter synaptic plasticity and contribute to αS misfolding that culminates in fatal neurodegeneration. 

Sequence and membrane determinants of the random coil-helix transition of α-synuclein.

Epithelially-derived ameloblasts secrete extracellular matrix proteins including amelogenin, enamelin, and ameloblastin. Complex intermolecular interactions among these proteins are believed to be important in controlling enamel formation. Here we provide in vitro and in vivo evidence of co-assembly and co-localization of ameloblastin with amelogenin using both biophysical and immunohistochemical methods. We performed co-localization studies using immunofluorescence confocal microscopy with paraffin-embedded tissue sections from mandibular molars of mice at 1, 5, and 8 days of age. Commercially-available ameloblastin antibody (M300) against mouse ameloblastin residues 107-407 and an antibody against full-length recombinant mouse (rM179) amelogenin were used. Ameloblastin-M300 clearly reacted along the secretory face of ameloblasts from days 1-8. Quantitative co-localization was analyzed (QCA) in several configurations by choosing appropriate regions of interest (ROIs). Analysis of ROIs along the secretory face of ameloblasts revealed that at day 1, very high percentages of both the ameloblastin and amelogenin co-localized. At day 8 along the ameloblast cells the percentage of co-localization remained high for the ameloblastin whereas co-localization percentage was reduced for amelogenin. Analysis of the entire thickness on day 8 revealed no significant co-localization of amelogenin and ameloblastin. With the progress of amelogenesis and ameloblastin degradation, there was a segregation of ameloblastin and co-localization with the C-terminal region decreased. CD spectra indicated that structural changes in ameloblastin occurred upon addition of amelogenin. Our data suggest that amelogenin-ameloblastin complexes may be the functional entities at the early stage of enamel mineralization. 

Analysis of co-assembly and co-localization of ameloblastin and amelogenin.

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.

Interactions of amelogenin with phospholipids.

We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed that they are structurally similar to the wild-type amelogenin. Utilizing the intrinsic fluorescence of single tryptophan residue and fluorescence resonance energy transfer [FRET], we analyzed the accessibility and strength of their binding with an ameloblast cell membrane-mimicking model membrane (ACML) and a negatively charged liposome used as a membrane model. We found that amelogenin has membrane-binding ability mainly via its N-terminal, close to residues W25 and W45. Significant blue shift was also observed in the fluorescence of a N-terminal peptide following addition of liposomes. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta (AI) with mutations at the N-terminal may be the result of defective amelogenin-cell interactions. 

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus.

Parkinson's disease (PD) is a neurodegenerative disorder involving progressive deterioration of dopaminergic neurons. Although few genetic markers for familial PD are known, the etiology of sporadic PD remains poorly understood. Microarray data was analysed for induced pluripotent stem cells (iPSCs) derived from PD patients and mature neuronal cells (mDA) differentiated from these iPSCs. Combining expression and semantic similarity, a highly-correlated PD interactome was constructed that included interactions of established Parkinson's disease marker genes. A novel three-way comparative approach was employed, delineating topologically and functionally important genes. These genes showed involvement in pathways like Parkin-ubiquitin proteosomal system (UPS), immune associated biological processes and apoptosis. Of interest are three genes, eEF1A1, CASK, and PSMD6 that are linked to PARK2 activity in the cell and thereby form attractive candidate genes for understanding PD. Network biology approach delineated in this study can be applied to other neurodegenerative disorders for identification of important genetic regulators.

Gene co-expression network analysis for identifying genetic markers in Parkinson's disease - a three-way comparative approach.

Parkinson's disease (PD) is a complex neurodegenerative movement disorder that primarily results due to the loss of dopaminergic neurons in the substantia nigra region. Studying gene expression in the substantia nigra region would potentially unravel disease-relevant protein-protein interactions.

Construction of Parkinson's disease marker-based weighted protein-protein interaction network for prioritization of co-expressed genes.

The organization of caveolae ultrastructures in the plasma membrane and the functions they dictate are mediated by membrane-embedded caveolins (caveolin-1, 2, 3) and peripherally attached cavins (cavin-1, 2, 3, 4). Mutations in caveolin and cavin genes are associated with a variety of human diseases. Cavin-1/PTRF mutations are known to contribute to several human pathologies, including muscular dystrophy and congenital generalized lipodystrophy (CGL). In the present study, we investigated the membrane interaction of the second leucine zipper domain (LZD2) of cavin-1 and the analogous peptide stretch in its CGL frameshift mutant (p.Glu176Argfs). The fluorescence data from the Trp-tagged peptides suggest binding of both wild-type and mutant peptide with negatively-charged membranes. The mutant peptide displayed a rather enhanced interaction compared to the wild-type peptide. In addition, the mutant peptide displayed appreciable binding to the lipid raft-mimicking cholesterol/sphingomyelin-rich vesicles as well. The alteration in the dynamics of peptide-lipid interaction is attributed to increased charge and hydrophilicity of the mutant peptides. Overall, these results suggest that the frameshift mutation in cavin-1/PTRF (p.Glu176Argfs) imparts high membrane-binding propensity to the region corresponding to LZD2, which is hitherto unknown to interact with membranes. Such interaction in the disease condition, in turn, could either alter the native membrane interaction dynamics of cavin-1/PTRF or possibly result in interaction with non-target membranes.

Interaction of cavin-1/PTRF leucine zipper domain 2 and its congenital generalized lipodystrophy mutant with model membranes.

Rhodopsin is a G-protein coupled receptor (GPCR) that mediates vision under dim light. Upon light exposure, rhodopsin is phosphorylated at multiple serine and threonine sites at its carboxyl-terminus by rhodopsin kinase (GRK1). This, in turn, reduces its ability to activate the visual G-protein transducin. Binding of light-activated, phosphorylated rhodopsin by arrestin (ARR1) fully terminates the catalytic activity of rhodopsin. Quantification of the levels of the differentially phosphorylated rhodopsin species provides definitive information about the role of phosphorylated rhodopsin in visual functions. Isoelectric Focusing (IEF) is a technique which is used to separate ampholytic components, such as proteins, based on their isoelectric point (pI). It is a useful technique used to distinguish protein isoforms and post-translational modifications such as phosphorylation, glycosylation, deamination, and acetylation, due to their effects on the protein's pI. Isoelectric Focusing can provide high resolution of differentially phosphorylated forms of a protein. Though other techniques such as kinase activity assays, phospho-specific antibodies, western blot, enzyme-linked immunosorbent assays (ELISA), radiolabeling and mass spectrometry are used to detect and quantify protein phosphorylation, IEF is a simple and cost-effective method to quantify rhodopsin phosphorylation, as it can readily detect individual phosphorylated forms. Here we provide a detailed protocol for determining phosphorylated rhodopsin species using the Isoelectric Focusing technique.

Sowmya Lokappa

Zilkha Neurogenetic Institute,
Keck School of Medicine,
Zilkha Neurogenetic Institute, Keck School of Medicine

University of Southern California

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Sowmya Lokappa

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University of Southern California

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Zilkha Neurogenetic Institute,
Keck School of Medicine,
Zilkha Neurogenetic Institute, Keck School of Medicine