Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

Fig. 4. 2D polyacrylamide gel electrophoresis (2D-PAGE) of retinal protein lysates. Coomassie Blue-stained gels and oxyblots are obtained by using 2D-PAGE of the equally loaded retinal protein samples from control or ocular hypertensive rat eyes. Protein carbonyl immunoreactivity detected on 2D-oxyblots, which reflects oxidatively modified proteins, occurs with a great extent in the retina of ocular hypertensive eyes (PI, isoelectric point). Arrows show the protein spots identified by using mass spectrometry, which included GAPDH, a glycolytic enzyme; HSP72, a stress protein; and glutamine synthetase, an excitotoxicity-related protein. This figure was modified with permission from the original (12).

enzyme; HSP72, a stress protein; and glutamine synthetase, an excitotoxicity-related protein (12).

By identifying retinal protein oxidation, this in vivo study supports the association of oxidative damage with glaucomatous neurodegeneration. In addition to the depletion of cellular redox-balance, protein oxidation may lead to loss in the specific function of many important retinal proteins in glaucomatous eyes, thereby leading to cell death. Because GAPDH, HSP72, and glutamine synthetase are known to play important roles for cell survival and/or function in the retina, their oxidative modification in the ocular hypertensive retina appears to have important implications in the neurodegenerative process of glaucoma (12). For example, oxidative inactivation of GAPDH in the ocular hypertensive retina may lead to impaired glucose utilization, critically important for RGC survival (34). Besides its pivotal glycolytic function, GAPDH is also involved in many other cellular processes, including apoptosis signaling (35,36). Therefore, oxidative modification of this multifunctional protein may also be important for the regulation of apoptosis signaling during glaucomatous neurodegeneration (35,37). Because appropriate expression of heat shock proteins is critical for their function in cellular protection, it is likely that alterations in HSP72 activity because of oxidative protein modification in ocular hypertensive eyes could ultimately

lead to impaired cellular response to tissue stress in these eyes (38). And, finally, glutamine synthetase is known to be particularly sensitive to inactivation by oxidants, and altered activity of this protein following oxidation has been shown to lead to the accumulation of glutamate resulting in neurotoxicity (39,40). Therefore, alteration in glutamine synthetase activity because of oxidative protein modification in ocular hypertensive eyes appears to be important in controlling the potential neurotoxicity of extracellular glutamate on RGCs.

Protein Phosphorylation

Given that phosphorylation is a key mechanism in intracellular signal transduction, identification of phosphorylated proteins can provide specific information with important implications for pathogenic mechanisms. Detection, identification, and quantification of phosphoproteins, as well as mapping of their phosphorylation sites, therefore constitute a major aim of proteomic studies. It is evident that phosphorylation cascades regulate the functional activity of many proteins involved in RGC signaling during glaucomatous neurodegeneration. Findings of in vitro (13) and histopathological (10) studies support that protein phosphorylation constitutes an important component of the cellular signaling determining RGC fate in glaucoma, because the functional activation of several adaptive/protective or pathogenic proteins is known to require phosphorylation. An improved understanding of phosphorylation cascades activated in RGCs can help therapeutic manipulation of RGC survival in glaucomatous eyes. In fact, information obtained from proteomics can directly contribute to drug development as almost all drugs are directed against proteins, and kinase signaling pathways have been a major focus of targeted therapeutics.

Protein phosphorylation can be effectively studied by MS, because phosphopeptides are 80 kDa heavier than their unmodified counterparts; they give rise to a specific fragment, are recognized by specific antibodies, bind to metal resins, and their phosphate groups can be removed by phosphatase treatment (41–44). For example, 2D-PAGE performed before and after phosphatase treatment can provide additional information, because protein phosphatase treatment inhibits the acidic shift consistent with protein phosphorylation. On the other hand, inhibition of intrinsic phosphatase activity is important to preserve phosphorylation states during phosphoprotein identification.

Because phosphorylation is a highly dynamic process, identification of phosphoproteins requires relatively more challenging approaches. To improve the sensitivity of phosphoprotein identification, various enrichment techniques are utilized. Through such strategies, low-abundance phosphoproteins that cannot be visualized and/or identified from the whole cell lysates can easily be enriched. In addition to 32P labeling, iron metal affinity chromatography and phosphopeptide-specific antibodies are most commonly used to enrich phosphopeptides for mass spectrometric analysis. Tandem MS has high sensitivity to identify phosphopeptides, eliminates possible signal suppression by non-phosphorylated peptides in the sample, and also allows the identification of phosphorylation sites through peptide sequencing and bioinformatics database searching (41–43). Gel-free LC/MS/MS analysis combined with multiple separate proteolytic digests yields higher sequence coverage for phosphoprotein identification.

Stable isotope labeling methods also facilitate relative quantification of phosphoproteins through LC/MS/MS analysis. Emerging improvements in MS techniques and availability of software algorithms are expected to further extend the capabilities and sensitivity of phosphoprotein identification (42,45,46).

As an initial proteomic approach to identify differential protein phosphorylation in the ocular hypertensive retina, phosphoprotein staining of 2D-gels has recently been utilized before protein identification by tandem MS (47). Through this approach, phosphorylated proteins can be directly stained on 2D-gels followed by Sypro Ruby staining for total protein profiling. Tandem MS then identifies the proteins revealed by the specific staining, which is fully compatible with MS (48). As shown in Fig. 5, comparison of Pro-Q Diamond-stained and Sypro Ruby-stained 2D-gels revealed that the number and intensity of protein spots exhibiting phosphorylation are greater in ocular hypertensive retinas compared with the controls (47). Among over a hundred spots exhibiting new or increased phosphorylation, the proteins identified with significant matches included those involved in cell signaling (collapsin response mediator proteins, 14-3-3 family proteins, dC stretch-binding protein, ubiquinol-cytochrome c reductase core protein, and annexin) and stress response (HSP60, HSP70, and matricin). Immunolabeling of retina sections with specific antibodies demonstrated the cellular localization of these proteins, which included RGCs. Findings of this in vivo study support that the identified proteins, most of which have been associated with other neurodegenerative injuries to the brain, may have important implications in pathogenic mechanisms of neurodegeneration in glaucoma. For example, collapsin response mediator proteins are involved in the regulation of axonal outgrowth and guidance (49,50). These proteins belong to the semaphorins family, which are key proteins modulating the cell fate of axotomized RGCs (51). In addition, hyperphosphorylation of brain collapsin response mediator proteins in Alzheimer’s disease has been associated with the neurodegenerative pathology (52,53). On the other hand, 14- 3-3 family of proteins is involved in the control of a wide range of vital regulatory

Fig. 5. 2D-PAGE of retinal protein lysates. Phosphorylated retinal proteins can be stained on 2D-gels using Pro-Q Diamond followed by Sypro Ruby staining for total protein profiling. The number and intensity of protein spots exhibiting phosphorylation were found greater in ocular hypertensive retinas compared with the controls. Mass spectrometry identified the proteins revealed by the specific staining, which included those involved in cell signaling (1, collapsin response mediator proteins; 2, 14-3-3 family proteins; 3, dC stretch-binding protein; 4, ubiquinol-cytochrome c reductase core protein; and 5, annexin) and stress response (6, HSP60; 7, HSP70; and 8, matricin).

processes, including apoptotic cell death. For example, these proteins bind and lead to cytoplasmic localization of Bad, thus preventing this proapoptotic protein from entering mitochondria to initiate apoptosis (54–56). 14-3-3 proteins also play an organizing role in the intermediate filament network in reactive astrocytes during neurodegeneration (57) and have been associated with the relative protection of astrocytes against ischemic injury (58). Further studies should help better understand the importance of these proteins in cellular processes and fully identify signaling cascades promoting RGC death during glaucomatous neurodegeneration.

Protein Glycation

A recent histopathological study has demonstrated an accelerated accumulation of advanced glycation end-products (AGEs) in the glaucomatous human retina and optic nerve head (59). Findings of this study support that the process of protein glycation, non-enzymatic glycosylation, is not limited to diabetic patients, but is also associated with glaucomatous neurodegeneration. Another common feature of glaucoma and diabetes is that RGC apoptosis, characteristic of glaucomatous neurodegeneration, is also detected in diabetic eyes. However, whether there are any overlapping mechanisms for such common damage to RGCs in these two distinct diseases is unclear. On the basis of the evidence of enhanced AGE accumulation in glaucomatous tissues similar to diabetes, a recent in vivo study aimed to determine whether protein glycation could be a common determinant of optic nerve neurodegeneration in glaucoma and diabetes (14). Utilizing experimental models of these diseases, retinal protein samples were obtained from rat eyes matched for IOP exposure and axon loss or blood glucose levels. During an experimental period of up to 12 weeks, glycated retinal proteins were detected by Pro-Q Emerald 300 glycoprotein staining of 2D-gels. For large-scale quantification, differential display analysis was performed to compare the intensity of retinal protein spots matched on 2D-gel images obtained from ocular hypertensive and diabetic eyes relative to controls. The number and intensity of protein spots exhibiting glycation were found to be greater in 2D-gels obtained using protein lysates from diabetic retinas. In contrast to controls, protein glycation was also detected in 2D-gels obtained using protein lysates of ocular hypertensive retinas at over 20 spots shared with diabetic retinas. The common glycated proteins revealed by the specific staining were identified using tandem MS. The proteins identified with significant matches, most of which have also been oxidized, included cytoskeletal proteins, -actin and-tubulin, as well as some glycolytic enzymes and signaling molecules. 2D-western blot analysis using specific antibodies confirmed the identified proteins. In addition, immunofluorescence microscopy and electron microscopy were utilized to determine cellular localization of the identified proteins in the retina and ultrastructural alterations associated with these proteins, respectively. Despite widespread cellular localization of -actin and -tubulin in retinal neurons and glia, electron microscopic findings were consistent with cytoskeletal alterations prominent in neurons of the ocular hypertensive and diabetic retinas, both including RGCs. In agreement with previous observations that tubulin glycation leads to the inhibition of tubulin polymerization, electron microscopy demonstrated amorphous aggregates of cytoskeleton without characteristic microtubular structures in these retinas.

Thus, the findings of this in vivo study support aberrant protein glycation in ocular hypertensive as well as diabetic retinas, and identify common targets of this posttranslational protein modification in two different disease models. Increased retinal protein glycation in ocular hypertensive rats is consistent with the evidence of AGE accumulation in ocular tissues of non-diabetic glaucoma patients, as well as the AGE accumulation by physiological aging. The process of AGE generation agrees that not only glucose but also various other sugars and their end-compounds can participate in the glycation of proteins (60,61). Such an aberrant protein glycation, also evident in other neurodegenerative diseases of the brain such as Alzheimer’s disease, is known to be accelerated by oxidative stress end-products (62) evident in glaucomatous eyes (12). Because glycation may interact with and even compete with phosphorylation, this protein modification may also interfere with cell signaling. In addition to structural alterations, glycation may also influence the biological activity of proteins by affecting their folding and stability. This has been associated with the impaired retrograde axonal transport in diabetic animals, which relies on the integrity of the axonal cytoskeleton. Similarly, glycation-associated structural and/or functional alterations in cytoskeletal integrity may commonly contribute to increased susceptibility of RGCs to damage in both glaucoma and diabetes. Besides neurons, glycation-associated dysfunction in glial cells may also lead to diminished glial support, thereby being another common factor facilitating the neuronal damage in both ocular hypertensive and diabetic retinas.

Identification of Protein Interactions During Neurodegenerative Signaling

In addition to post-translational modification of proteins, protein interactions can also be identified by profiling specific multi-protein complexes in such a way that downstream signaling complexes can be isolated and identified step-by-step through the specific signaling pathways. Several proteomic approaches have been successfully used to identify protein–protein interactions involved in intracellular signaling with a higher success rate compared with two-hybrid studies (32,33,63).

Ongoing studies using the experimental model of glaucomatous neurodegeneration currently isolate and purify multi-protein complexes through antibody-based co-immunoprecipitation and recombinant protein-based affinity pull-down. These two techniques, when used in parallel, can provide complementary and confirmatory information about signaling complexes. Antibody-based co-immunoprecipitation targets preexisting multi-protein complexes using an antibody against one of the components. This method isolates the complexes without disrupting them but is limitedin its ability to identify specific complexes in which the antibody-directed epitope may be masked by interacting proteins. Additionally, the antibody-binding event may displace proteins that interact with the epitope region of the target protein, and hence, these interacting proteins may escape the analysis. In contrast, recombinant protein-based affinity pull-down introduces exogenous full-length proteins into the in vitro system (cell lysate). In this assay, the bait protein competes with the endogenous protein to form a new multi-protein complex. This method is advantageous because epitope tagging of the recombinant protein facilitates the pull-down and reduces the likelihood that the epitope for isolation is masked. Many low-abundance proteins of a variety of functional classes, including kinases and their targets, can be identified by this

single-step purification procedure, without the risk of losing some less stable interactions. These procedures followed by mass spectrometric identification of the associated proteins have been increasingly used to identify protein–protein interactions involved in intracellular signaling (32,63). These approaches of targeted proteomics can also identify proteins in a variety of concentrated sub-cellular compartments of interest, such as the plasma membrane, nucleus, cytoplasm, or mitochondrion. Examination of subcellular compartments can provide additional information, because protein complexes may be compartmentalized at targeted sub-cellular locations, or organelle-specific structural proteins may be associated with cellular signaling (33).

Once interacting proteins are identified and a specific pathway map is obtained, loss-of-function analysis can be performed using the RNA interference technology to obtain a comprehensive functional map of a pathogenic pathway and determine therapeutically relevant signaling molecules (64). Single or multiple perturbation with small-interfering RNAs mimics pharmacological treatment and can therefore be used to model the effects of inhibiting drug targets and to choose the most effective intervention strategy.

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