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: (i) the levels of inflammation-associated proteins such as alpha1-antitrypsin, apolipoprotein A4, albumin, and transferrin were significantly higher in all four types of vitreoretinal diseases, and (ii) each vitreoretinal disease elevated 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 properties.

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.

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The gene encoding the 49-kilodalton protein that undergoes light-induced phosphorylation in the Drosophila photoreceptor has been isolated and characterized. The encoded protein has 401 amino acid residues and a molecular mass of 44,972 daltons, and it shares approximately 42 percent amino acid sequence identity with arrestin (S-antigen), which has been proposed to quench the light-induced cascade of guanosine 3',5'-monophosphate hydrolysis in vertebrate photoreceptors. Unlike the 49-kilodalton protein, however, arrestin, which appears to bind to phosphorylated rhodopsin, has not itself been reported to undergo phosphorylation. In vitro, Ca2+ was the only agent found that would stimulate the phosphorylation of the 49-kilodalton protein. The phosphorylation of this arrestin-like protein in vivo may therefore be triggered by a Ca2+ signal that is likely to be regulated by light-activated phosphoinositide-specific phospholipase C.

Interaction between the ␥ subunit (P␥) of cGMP phosphodiesterase and the ␣ subunit (T␣) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an endogenous enzyme and site-directed mutagenesis of the P␥ polycationic region to identify residues required for the interaction with T␣. P␥, free or complexed with the ␣␤ subunit (P␣␤) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32 P]NAD. Identification of ADP-ribose in the radiolabeled P␥ and radiolabeling of arginine-replaced mutant forms of P␥ indicate that both arginine 33 and arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is modified at a time. P␥ complexed with T␣ (both GTP-and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with T␣, was ADP-ribosylated in the presence of T␣, suggesting that a P␥ domain containing these arginines is masked by T␣. A P␥ mutant (R33,36K), as well as wild type P␥, inhibited both GTP hydrolysis of T␣ and GTP binding to T␣. Moreover, GTP-bound T␣ activated P␣␤ that had been inhibited by R33,36K. However, another P␥ mutant (R33,36L) could not inhibit these T␣ functions. In addition, GTP-bound T␣ could not activate P␣␤ inhibited by R33,36L. These results indicate that a P␥ domain containing these arginines is required for its interaction with T␣, but not with P␣␤, and that positive charges in these arginines are crucial for the interaction.Cyclic GMP phosphodiesterase (PDE), 1 a key enzyme in phototransduction, is composed of P␣␤ and two P␥ subunits (1-6). P␣␤ hydrolyzes cGMP (7,8) and binds cGMP to its high affinity, noncatalytic sites (9 -11). In amphibian ROS, P␥ regulates these P␣␤ functions as an inhibitor of cGMP hydrolysis (12) and as a stimulator of cGMP binding to noncatalytic sites (13,14). Different interactions between P␣␤ and P␥ have been suggested to be required to express these two functions (15, 16). In bovine ROS, P␥ inhibits cGMP hydrolysis by P␣␤ (17); however, the effect of P␥ on the cGMP binding to noncatalytic sites has never been documented. In amphibian ROS, these P␥ functions are interrupted by P␥ release with GTP⅐T␣ from P␣␤ (12-14, 18). We have recently suggested that these functionally different P␥s are released in the different steps of phototransduction (15, 16). When [cGMP] is at the dark level, P␥ responsible for the inhibition of cGMP hydrolysis is released. Consequently, cGMP is hydrolyzed by the activated PDE for photoexcitation. When [cGMP] becomes low, P␥ responsible for the stimulation of cGMP binding is released, and the affinities of these noncatalytic sites to cGMP are drastically reduced. The resulting release of cGMP from these noncatalytic sites may facilitate the recovery of cytoplasmic [cGMP] to the dark level in ROS.To understand physiological functions of protein-protein interaction, functional structures of the protein i...