Exploration of cone cyclic nucleotide-gated channel-interacting proteins using affinity purification and mass spectrometry.

Photopic (cone) vision essential for color sensation, central vision, and visual acuity is mediated by the activation of photoreceptor cyclic nucleotide-gated (CNG) channels. Naturally occurring mutations in the cone channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. This work investigated the functional modulation of cone CNG channel by exploring the channel-interacting proteins. Retinal protein extracts prepared from cone-dominant Nrl (- / -) mice were used in CNGA3 antibody affinity purification, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The peptide mass fingerprinting of the tryptic digests and database search identified a number of proteins including spectrin alpha-2, ATPase (Na(+)/K(+) transporting) alpha-3, alpha and beta subunits of ATP synthase (H(+) transporting, mitochondrial F1 complex), and alpha-2 subunit of the guanine nucleotide-binding protein. In addition, the affinity-binding assays demonstrated an interaction between cone CNG channel and calmodulin but not cone Na(+)/Ca(2+)-K(+) exchanger in the mouse retina. Results of this study provide insight into our understanding of cone CNG channel-interacting proteins and the functional modulations.

Retinophilin is a light-regulated phosphoprotein required to suppress photoreceptor dark noise in Drosophila.

Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking neither inactivation nor afterpotential C (NINAC) myosin III, a motor protein/kinase, also display a similar dark-noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is attributable to lack of RTP and, furthermore, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors.

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 2: isolation and characterization of the transducin-activated form.

Rod photoreceptor cGMP phosphodiesterase (PDE6) consists of a catalytic subunit complex (Palphabeta) and two inhibitory subunits (Pgamma). In the accompanying article, using bovine photoreceptor outer segment homogenates, we show that Pgamma as a complex with the GTP-bound transducin alpha subunit (GTP-Talpha) dissociates from Palphabetagammagamma on membranes, and the Palphabetagammagamma becomes Pgamma-depleted. Here, we identify and characterize the Pgamma-depleted PDE. After incubation with or without guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), Palphabeta complexes are extracted. When a hypotonic buffer is used, Palphabetagammagamma, Palphabetagamma, and a negligible amount of a Palphabeta complex containing Pgamma are isolated with GTPgammaS, and only Palphabetagammagamma is obtained without GTPgammaS. When an isotonic buffer containing Pdelta, a prenyl-binding protein, is used, Palphabetagammagammadelta, Palphabetagammadeltadelta, and a negligible amount of a Palphabeta complex containing Pgamma and Pdelta are isolated with GTPgammaS, and Palphabetagammagammadelta is obtained without GTPgammaS. Neither Palphabeta nor Palphabetagammagamma complexed with GTPgammaS-Talpha is found under any condition we examined. Palphabetagamma has approximately 12 times higher PDE activity and approximately 30 times higher Pgamma sensitivity than those of Palphabetagammagamma. These results indicate that the Pgamma-depleted PDE is Palphabetagamma. Isolation of Palphabetagammagammadelta and Palphabetagammadeltadelta suggests that one C-terminus of Palphabeta is involved in the Palphabetagammagamma interaction with membranes, and that Pgamma dissociation opens another C-terminus for Pdelta binding, which may lead to the expression of high PDE activity. Cone PDE behaves similarly to rod PDE in the anion exchange column chromatography. We conclude that the mechanisms for PDE activation are similar in mammalian and amphibian photoreceptors as well as in rods and cones.

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: identification of its inhibitory subunit complexes and their roles.

Cyclic GMP phosphodiesterase (PDE) in bovine rod photoreceptor outer segments (OS) comprises a catalytic subunit complex (Palphabeta) and two inhibitory subunits (Pgamma) and is regulated by the alpha subunit of transducin (Talpha). Here, we show an overall mechanism for PDE regulation by identifying Pgamma complexes in OS homogenates prepared with an isotonic buffer. Before Talpha activation, three Pgamma complexes exist in the soluble fraction. Complex a, a minor complex, contains Palphabeta, Talpha, and a protein named Pdelta. Complex b, Palphabetagammagamma( b ), has a PDE activity similar to that of membranous Palphabetagammagamma, Palphabetagammagamma( M ), and its level, although its large portion is Pdelta-free, is estimated to be 20-30% of the total Palphabetagammagamma. Complex c, (Pgamma.GDP-Talpha) (2) ( c ) , appears to be a dimer of Pgamma.GDP-Talpha. Upon Talpha activation, (1) complex a stays unchanged, (2) Palphabetagammagamma( b ) binds to membranes, (3) the level of (Pgamma.GDP-Talpha) (2) ( c ) is reduced as its GTP-form is produced, (4) complex d, Pgamma.GTP-Talpha( d ), is formed on membranes and its substantial amount is released to the soluble fraction, and (5) membranous Palphabetagammagamma, Palphabetagammagamma( M ) and/or Palphabetagammagamma( b ), becomes Pgamma-depleted. These observations indicate that Pgamma as a complex with GTP-Talpha dissociates from Palphabetagammagamma on membranes and is released to the soluble fraction and that Pgamma-depleted PDE is the GTP-Talpha-activated PDE. After GTP hydrolysis, both (Pgamma.GDP-Talpha) (2) ( c ) and Pgamma.GDP-Talpha( d ), without liberating Pgamma, deactivate Pgamma-depleted PDE. The preferential order to be used for the deactivation is membranous Pgamma.GDP-Talpha( d ), solubilized Pgamma.GDP-Talpha( d ) and (Pgamma.GDP-Talpha) (2) ( c ) . Release of Pgamma.GTP-Talpha complexes to the soluble fraction is relevant to light adaptation.

Determination of Protein Molecular Weights on SDS-PAGE.

An apparent molecular weight (MW) of a protein can be determined from the migration distance of a protein complexed with a strong cationic detergent sodium dodecyl sulfate (SDS) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This method was established around 1969 and has been utilized substantially even today because of its simplicity. During the following half a century, although it has been reported that many proteins show some deviation in MW when determined on SDS-PAGE especially when their peptide chains are posttranslationally modified, this versatile method is still being used very often in current biochemical works. In this protocol, a simple method to estimate MW by running SDS-PAGE of standard proteins is explained by an example in which proteins extracted from mouse retina were analyzed by two-dimensional isoelectric focusing (2-D IEF) SDS-PAGE followed by protein identification by peptide mass fingerprinting.

Identification of Proteins on Archived 2D Gels.

Separation technology of proteins from a complex mixture by two-dimensional gel (2D gel) was invented more than 40 years ago. With a good laboratory practice, the 2D gels are likely to be dried and stored at ambient temperature as archived record. Up until the beginning of this century, it had been difficult to identify the protein spots isolated on 2D gels. However, the advent of mass spectrometry-based proteomics protocols combined with genome information enabled us to determine the identity of a protein separated on 2D gels archived decades ago. The protocol will assist researchers to decipher molecular mechanisms involved in the system by identifying and quantifying the protein of interest from archived 2D gels.

Two-Dimensional Gel Electrophoresis by Glass Tube-Based IEF and SDS-PAGE.

The genome information combined with data derived from modern mass spectrometry enables us to determine the identity of a protein once it is isolated from a complex mixture. Two-dimensional gel electrophoresis established more than four decades ago serves as a powerful protocol to isolate many proteins at once for such protein analysis. In the first two decades, the original procedure to use a glass tube-based IEF had been commonly used. Since an IEF in glass tubes is rather difficult to maneuver, a new method to use an IEF on a thin agarose slab backed by a plastic film (IPG Dry Strip) had been invented and is now widely used. In this chapter, we describe a protocol that uses a glass tube-based IEF because the capacity of protein loading and resolving power of this type of classic two-dimensional gel is still indispensable for many applications, not only for protein identification but also for protocols that are benefited by larger amounts of materials, i.e., analysis of posttranslational modification of proteins such as phosphorylation, methylation, glycosylation, and others.

Extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) pathways involved in spinal cord stimulation (SCS)-induced vasodilation.

BACKGROUND AND AIMS: SCS is used to improve peripheral circulation in selected patients with ischemia of the extremities. However the mechanisms are not fully understood. The present study investigated whether blockade of ERK and AKT activation modulated SCS-induced vasodilation. METHODS: A unipolar ball electrode was placed on the left dorsal column at the lumbar 2-3 spinal segments in rats. Cutaneous blood flows from left and right hind foot pads were recorded with laser Doppler flow perfusion monitors. SCS was applied through a ball electrode at 60% or 90% of MT. U0126, an inhibitor of ERK kinase, or LY294002, an inhibitor of PI3K upstream of AKT, was applied to the lumbar 3-5 spinal segments (n=7, each group). RESULTS: U0126 (100 nM, 5 microM and 250 microM) significantly attenuated SCS-induced vasodilation at 60% (100 nM: P<0.05; 5 microM and 250 microM: P<0.01, respectively) and 90% of MT (100 nM and 5 microM: P<0.05; 250 microM: P<0.01, respectively). LY294002 at 100 microM also attenuated SCS-induced vasodilation at 60% and 90% of MT (P<0.05). CONCLUSIONS: These data suggest that ERK and AKT pathways are involved in SCS-induced vasodilation.

Proteomics study of neuropathic and nonneuropathic dorsal root ganglia: altered protein regulation following segmental spinal nerve ligation injury.

Peripheral nerve injury is often followed by the development of severe neuropathic pain. Nerve degeneration accompanied by inflammatory mediators is thought to play a role in generation of neuropathic pain. Neuronal cell death follows axonal degeneration, devastating a vast number of molecules in injured neurons and the neighboring cells. Because we have little understanding of the cellular and molecular mechanisms underlying neuronal cell death triggered by nerve injury, we conducted a proteomics study of rat 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) after L5 spinal nerve ligation. DRG proteins were displayed on two-dimensional gels and analyzed through quantitative densitometry, statistical validation of the quantitative data, and peptide mass fingerprinting for protein identification. Among approximately 1,300 protein spots detected on each gel, we discovered 67 proteins that were tightly regulated by nerve ligation. We find that the injury to primary sensory neurons turned on multiple cellular mechanisms critical for the structural and functional integrity of neurons and for the defense against oxidative damage. Our data indicate that the regulation of metabolic enzymes was carefully orchestrated to meet the altered energy requirement of the DRG cells. Our data also demonstrate that ligation of the L5 spinal nerve led to the upregulation in the L4 DRG of the proteins that are highly expressed in embryonic sensory neurons. To understand the molecular mechanisms underlying neuropathic pain, we need to comprehend such dynamic aspect of protein modulations that follow nerve injury.

Roles of peripheral terminals of transient receptor potential vanilloid-1 containing sensory fibers in spinal cord stimulation-induced peripheral vasodilation.

BACKGROUND: Spinal cord stimulation (SCS) is used to relieve ischemic pain and improve peripheral blood flow in selected patients with peripheral arterial diseases. Our previous studies show that antidromic activation of transient receptor potential vanilloid-1 (TRPV1) containing sensory fibers importantly contributes to SCS-induced vasodilation. OBJECTIVES: To determine whether peripheral terminals of TRPV1 containing sensory fibers produces vasodilation that depends upon the release of calcitonin gene-related peptide (CGRP) and nitric oxide (NO) during SCS. METHODS: A unipolar ball electrode was placed on the left dorsal column at lumbar spinal cord segments 2-3 in sodium pentobarbital anesthetized, paralyzed and ventilated rats. Cutaneous blood flow from left and right hindpaws was recorded with laser Doppler flow perfusion monitors. SCS was applied through a ball electrode at 30%, 60%, 90% and 300% of motor threshold. Resiniferatoxin (RTX; 2 microg/ml, 100 microl), an ultra potent analog of capsaicin, was injected locally into the left hindpaw to functionally inactivate TRPV-1 containing sensory terminals. In another set of experiments, CGRP(8-37), an antagonist of the CGRP-1 receptor, was injected at 0.06, 0.12 or 0.6 mg/100 microl into the left hindpaw to block CGRP responses; N-omega-nitro-l-arginine methyl ester (L-NAME), a nonselective nitric-oxide synthase (NOS) inhibitor, was injected at 0.02 or 0.2 mg/100 microl into the left hindpaw to block nitric oxide synthesis; (4S)-N-(4-Amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine, TFA, a neuronal NOS inhibitor, was injected at 0.02 or 0.1 mg/100 microl into the left hindpaw to block neuronal nitric oxide synthesis. RESULTS: SCS at all intensities produced vasodilation in the left hindpaw, but not in the right. RTX administration attenuated SCS-induced vasodilation at all intensities in the left hindpaw (P<0.05, n=7) compared with responses before RTX. CGRP(8-37) administration attenuated SCS-induced vasodilation in the left hindpaw in a dose dependent manner (linear regression, P<0.05) compared with responses before CGRP(8-37). In addition, L-NAME at a high dose, but not (4S)-N-(4-Amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine, TFA, decreased SCS-induced vasodilation (P<0.05, n=5). CONCLUSION: While TRPV1, CGRP and NO are known to be localized in the same nerve terminals, our data indicate that SCS-induced vasodilation depends on CGRP release, but not NO release. NO, released from endothelial cells, may be associated with vascular smooth muscle relaxation and peripheral blood flow increase in response to SCS.

Sensory fibers containing vanilloid receptor-1 (VR-1) mediate spinal cord stimulation-induced vasodilation.

BACKGROUND AND AIMS: Spinal cord stimulation (SCS) is used to improve peripheral blood flow in selected populations of patients with ischemia of the extremities. Previous studies show that antidromic activation of sensory fibers is an important mechanism that contributes to SCS-induced vasodilation. However, the characteristics of sensory fibers involved in vasodilation are not fully known. This study investigated the contribution of vanilloid receptor type 1 (VR-1) containing fibers to SCS-induced vasodilation. METHODS: A unipolar ball electrode was placed on the left dorsal column at the lumbar 2-3 spinal cord segments (L2-L3) in sodium pentobarbital anesthetized, paralyzed and ventilated rats. Cutaneous blood flows from both ipsilateral (left) and contralateral (right) hind foot pads were recorded with laser Doppler flow perfusion monitors. SCS (50 Hz; 0.2 ms) was applied through the ball electrode at 30%, 60%, 90% and 300% of motor threshold (MT). Resiniferatoxin (RTX), an ultra potent analog of capsaicin and VR-1 receptor agonist, was used to suppress the activities of VR-1 containing sensory fibers. RESULTS: SCS at 30%, 60%, 90% and also at 300% of MT significantly increased cutaneous blood flow in the ipsilateral foot pad compared to that in the contralateral side. RTX (2 microg/kg, i.v.) significantly attenuated SCS-induced vasodilation of the ipsilateral side (P<0.05, n=7) compared with responses prior to RTX administration. A pledget of cotton soaked with RTX (2 microg/ml) placed on L2-L3 spinal cord significantly decreased SCS-induced vasodilation of the ipsilateral side at 30%, 60%, 90% and 300% of MT (P<0.05, n=7) compared with responses prior to RTX administration. Additionally, topical application of a pledget of cotton soaked with RTX (2 microg/ml) on the sciatic nerve at the middle level of the thigh or on the tibial nerve at the lower level of the lower hindlimb also decreased SCS-induced vasodilation (n=5). CONCLUSION: SCS-induced vasodilation is predominantly mediated via VR-1 containing sensory fibers.

Presence of beta-arrestin-1 immunoreactivity in the cutaneous nerve fibers of rat glabrous skin.

beta-Arrestin-1 (betaArr1) plays a major role in the desensitization and internalization of G protein-coupled receptors. We previously localized betaArr1 in the sensory neurons of rat lumbar 4 and 5 dorsal root ganglia (DRG) and reported the predominant presence of betaArr1 in the small-diameter DRG neurons that are often implicated with nociception. Because of betaArr1's crucial role in regulating the initiation of cellular signaling, in the current study we evaluated the distribution of betaArr1 in the peripheral sensory terminals where various receptors are present. Western blotting confirmed the presence of betaArr1 immunoreactivity in the rat skin. Sciatic nerve ligation demonstrated that betaArr1 is transported peripherally from the DRG, and immunohistochemistry showed betaArr1 immunoreactivity in the glabrous skin of the rat hindpaw. In the glabrous skin, strong betaArr1 immunoreactivity was detected in nerve fibers in the dermal nerve plexus and dermal papillae. Fine varicose immunoreactive fibers were found in the epidermis. In addition, betaArr1 was observed in specialized sensory receptors such as Meissner corpuscles. Our observations thus indicate that betaArr1 may be involved in modulation of specific tactile stimulation from the skin in addition to nociception.

Mechanisms of sustained cutaneous vasodilation induced by spinal cord stimulation.

This study was performed to investigate whether spinal cord stimulation (SCS) at intensities below motor threshold prolongs cutaneous vasodilation and whether sustained vasodilation by SCS is mediated through sympathetic inhibition and/or antidromic activation of sensory fibers. SCS was applied to the dorsal surface of the L2-L3 spinal cord of anesthesized rats with stimulus parameters used clinically (i.e., 50 Hz, 0.2 ms duration, and stimulus intensity at 30%, 60%, or 90% of motor threshold). Peripheral vasodilation induced by 5-min SCS was not attenuated by hexamethonium, an autonomic ganglion-blocking agent, but was abolished by dorsal rhizotomy. SCS at < or = 60% of motor threshold increased cutaneous blood flow to the level similar to that obtained at 90% of motor threshold, but the vasodilation did not last for 5 min. SCS-induced vasodilation at 90% of motor threshold persisted for the entire stimulation period up to 30 min, and the vasodilation was not attenuated by hexamethonium. It is concluded that sustained vasodilation, which is induced by SCS at only 90% of motor threshold, in this study was mediated via antidromic activation of sensory fibers.

Protein identification on archived 2-D gels.

Because of the availability of genome information combined with proteomics techniques, it is possible to determine the identity of a protein which had been isolated many years ago on a two-dimensional gel electrophoresis and stored in a dry state as a data archive. The protocol described in this chapter will assist researchers who want to know the identity of a protein separated decades ago when no techniques were available to determine the identity of the protein.

Two-dimensional gel electrophoresis: glass tube-based IEF followed by SDS-PAGE.

The genome information combined with data derived from modern mass spectrometry enables us to determine the identity of a protein once it is isolated from a complex mixture. Two-dimensional gel electrophoresis established more than three decades ago serves as a powerful protocol to isolate many proteins at once for such protein analysis. In the first two decades, the original procedure to use a glass tube-based isoelectric focusing (IEF) had been commonly used. Since an IEF in glass tubes is rather difficult to maneuver, a new method to use an IEF on a thin agarose slab backed by a plastic film (IPG Dry Strip) has been invented and is now widely used. In this chapter, we describe the original protocol that uses a glass tube-based IEF because, the capacity of protein loading and resolving power of this type of classic two-dimensional gel is still indispensible.

Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development.

Periodontal disease, gingival inflammation (gingivitis) and periodontal attachment loss (periodontitis), causes tooth loss and susceptibility to chronic inflammation. Professionally scaling and cleaning the teeth regularly controls the disease, but is expensive in companion animals. Eikenella corrodens is common in canine oral cavities where it is a source of lysine decarboxylase (LDC). In human dental biofilms (plaques), LDC converts lysine to cadaverine and impairs the gingival epithelial barrier to bacteria. LDC vaccination may therefore retard gingivitis development. Year-old beagle dogs provided blood samples, and had weight and clinical measurements (biofilm and gingivitis) recorded. After scaling and cleaning, two dogs were immunized subcutaneously with 0.2mg native LDC from E. corrodens and 2 sets of four dogs with 0.2mg recombinant LDC purified from Escherichia coli. A third set of 4 dogs was immunized intranasally. Rehydragel(®), Emulsigen(®), Polygen™ or Carbigen™ were used as adjuvant. Four additional pairs of dogs were sham-immunized with each adjuvant alone (controls). Immunizations were repeated twice, 3 weeks apart, and clinical measurements were obtained after another 2 weeks, when the teeth were scaled and cleaned again. Tooth brushing was then stopped and the diet was changed from hard to soft chow. Clinical measurements were repeated after 1, 2, 3, 4, 6 and 8 weeks. Compared with sham-immunized dogs, gingivitis was reduced over all 8 weeks of soft diet after subcutaneous immunization with native LDC, or after intranasal immunization with recombinant LDC in Carbigen™, but for only 6 of the 8 weeks after subcutaneous immunization with recombinant LDC in Emulsigen(®) (repeated measures ANOVA). Subcutaneous vaccination induced a strong serum IgG antibody response that decreased during the soft diet period, whereas intranasal immunization induced a weak serum IgA antibody response that did not decrease. Immunization with recombinant LDC may provide protection from gingivitis if procedures are optimized.

Proteome Profiling of Vitreoretinal Diseases by Cluster Analysis.

Vitreous samples collected in retinopathic surgeries have diverse properties, making proteomics analysis difficult. We report a cluster analysis to evade this difficulty. Vitreous and subretinal fluid samples were collected from 60 patients during surgical operation of non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy, and rhegmatogenous retinal detachment. For controls we collected vitreous fluid from patients of idiopathic macular hole, epiretinal, and from a healthy postmortem donor. Proteins from these samples were subjected to quantitative proteomics using two-dimensional gel electrophoresis. We selected 105 proteins robustly expressed among ca 400 protein spots and subjected them to permutation test. By using permutation test analysis we observed unique variations in the expression of some of these proteins in vitreoretinal diseases when compared to the control and to each other: 1) the levels of inflammation-associate proteins such as AAT, APOA4, ALB, and TF were significantly higher in all four types of vitreoretinal diseases, and 2) each vitreoretinal disease elevates a unique set of proteins which can be interpreted based on the pathology of retinopathy. Our protocol will be effective for the study of protein expression in other types of clinical samples of diverse property.

Novel eye-specific calmodulin methylation characterized by protein mapping in Drosophila melanogaster.

Post-translational methylation of the epsilon-amino group of lysine residues regulates a number of protein functions. Calmodulin, a key modulator of intracellular calcium signaling, is methylated on lysine 115 in many species. Although the amino acid sequence of calmodulin is highly conserved in eukaryotes, it has been shown that lysine 115 is not methylated in Drosophila calmodulin and no other methylation site has been reported. In this study, we characterized in vivo modification states of Drosophila calmodulin using proteomic methodology involving the protein mapping of microdissected Drosophila tissues on 2-D gels. We found that Drosophila calmodulin was highly expressed in methylated forms in the compound eye, whereas its methylation was hardly detected in other tissues. We identified that lysine 94 located in an EF-hand III is the methylation site in Drosophila calmodulin. The predominance of methylated calmodulin in the compound eye may imply the involvement of calmodulin in photoreceptor-specific functions through methylation.