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      Synthetic Polyclonal-Derived CDR Peptides as an Innovative Strategy in Glaucoma Therapy

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          Abstract

          The pathogenesis of glaucoma is strongly associated with the occurrence of autoimmune-mediated loss of retinal ganglion cells (RGCs) and additionally, recent evidence shows that specific antibody-derived signature peptides are significantly differentially expressed in sera of primary-open angle glaucoma patients (POAG) compared to healthy controls. Synthetically antibody-derived peptides can modulate various effector functions of the immune system and act as antimicrobial or antiviral molecules. In an ex vivo adolescent glaucoma model, this study, for the first time, demonstrates that polyclonal-derived complementarity-determining regions (CDRs) can significantly increase the survival rate of RGCs ( p = 0.013). We subsequently performed affinity capture experiments that verified the mitochondrial serine protease HTRA2 (gene name: HTRA2) as a high-affinity retinal epitope target of CDR1 sequence motif AS GYTFTNYGLSWVR. Quantitative proteomic analysis of the CDR-treated retinal explants revealed increased expression of various anti-apoptotic and anti-oxidative proteins (e.g., VDAC2 and TXN) compared to untreated controls ( p < 0.05) as well as decreased expression levels of cellular stress response markers (e.g., HSPE1 and HSP90AA1). Mitochondrial dysfunction, the protein ubiquitination pathway and oxidative phosphorylation were annotated as the most significantly affected signaling pathways and possibly can be traced back to the CDR-induced inhibition or modulation of the master regulator HTRA2. These findings emphasize the great potential of synthetic polyclonal-derived CDR peptides as therapeutic agents in future glaucoma therapy and provide an excellent basis for affinity-based biomarker discovery purposes.

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          Most cited references 77

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          Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.

          To characterize Brn3a expression in adult albino rat retinal ganglion cells (RGCs) in naïve animals and in animals subjected to complete intraorbital optic nerve transection (IONT) or crush (IONC). Rats were divided into three groups, naïve, IONT, and IONC. Two-, 5-, 9-, or 14-day postlesion (dpl) retinas were examined for immunoreactivity for Brn3a. Before the injury, the RGCs were labeled with Fluorogold (FG; Fluorochrome, Corp. Denver, CO). Brn3a retinal expression was also determined by Western blot analysis. The proportion of RGCs double labeled with Brn3a and FG was determined in radial sections. The temporal course of reduction in Brn3a(+) RGCs and FG(+) RGCs induced by IONC or IONT was assessed by quantifying, in the same wholemounts, the number of surviving FG-labeled RGCs and Brn3a(+)RGCs at the mentioned time points. The total number of FG(+)RGCs was automatically counted in naïve and injured retinas (2 and 5 dpl) or estimated by manual quantification in retinas processed at 9 and 14 dpl. All Brn3a immunopositive RGCs were counted using an automatic routine specifically developed for this purpose. This protocol allowed, as well, the investigation of the spatial distribution of these neurons. Brn3a(+) cells were only present in the ganglion cell layer and showed a spatial distribution comparable to that of FG(+) cells. In the naïve retinal wholemounts the mean (mean +/- SEM; n = 14) total number of FG(+)RGCs and Brn3a(+)RGCs was 80,251 +/- 2,210 and 83,449 +/- 4,541, respectively. Whereas in the radial sections, 92.2% of the FG(+)RGCs were also Brn3a(+), 4.4% of the RGCs were Brn3a(+)FG(-) and 3.4% were FG(+)Brn3a(-). Brn3a expression pattern was maintained in injured RGCs. The temporal course of Brn3a(+)RGC and FG(+)RGC loss induced by IONC or IONT followed a similar trend, but Brn3a(+)RGCs loss was detected earlier than that of FG(+)RGCs. Independent of the marker used to detect the RGCs, it was observed that their loss was quicker and more severe after IONT than after IONC. Brn3a can be used as a reliable, efficient ex vivo marker to identify and quantify RGCs in control and optic nerve-injured retinas.
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            VDAC2 inhibits BAK activation and mitochondrial apoptosis.

            The multidomain proapoptotic molecules BAK or BAX are required to initiate the mitochondrial pathway of apoptosis. How cells maintain the potentially lethal proapoptotic effector BAK in a monomeric inactive conformation at mitochondria is unknown. In viable cells, we found BAK complexed with mitochondrial outer-membrane protein VDAC2, a VDAC isoform present in low abundance that interacts specifically with the inactive conformer of BAK. Cells deficient in VDAC2, but not cells lacking the more abundant VDAC1, exhibited enhanced BAK oligomerization and were more susceptible to apoptotic death. Conversely, overexpression of VDAC2 selectively prevented BAK activation and inhibited the mitochondrial apoptotic pathway. Death signals activate "BH3-only" molecules such as tBID, BIM, or BAD, which displace VDAC2 from BAK, enabling homo-oligomerization of BAK and apoptosis. Thus, VDAC2, an isoform restricted to mammals, regulates the activity of BAK and provides a connection between mitochondrial physiology and the core apoptotic pathway.
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              The Structural Basis of Antibody-Antigen Recognition

              The function of antibodies (Abs) involves specific binding to antigens (Ags) and activation of other components of the immune system to fight pathogens. The six hypervariable loops within the variable domains of Abs, commonly termed complementarity determining regions (CDRs), are widely assumed to be responsible for Ag recognition, while the constant domains are believed to mediate effector activation. Recent studies and analyses of the growing number of available Ab structures, indicate that this clear functional separation between the two regions may be an oversimplification. Some positions within the CDRs have been shown to never participate in Ag binding and some off-CDRs residues often contribute critically to the interaction with the Ag. Moreover, there is now growing evidence for non-local and even allosteric effects in Ab-Ag interaction in which Ag binding affects the constant region and vice versa. This review summarizes and discusses the structural basis of Ag recognition, elaborating on the contribution of different structural determinants of the Ab to Ag binding and recognition. We discuss the CDRs, the different approaches for their identification and their relationship to the Ag interface. We also review what is currently known about the contribution of non-CDRs regions to Ag recognition, namely the framework regions (FRs) and the constant domains. The suggested mechanisms by which these regions contribute to Ag binding are discussed. On the Ag side of the interaction, we discuss attempts to predict B-cell epitopes and the suggested idea to incorporate Ab information into B-cell epitope prediction schemes. Beyond improving the understanding of immunity, characterization of the functional role of different parts of the Ab molecule may help in Ab engineering, design of CDR-derived peptides, and epitope prediction.
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                Author and article information

                Journal
                J Clin Med
                J Clin Med
                jcm
                Journal of Clinical Medicine
                MDPI
                2077-0383
                15 August 2019
                August 2019
                : 8
                : 8
                Affiliations
                Department of Experimental and Translational Ophthalmology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany
                Author notes
                [* ]Correspondence: grus@ 123456eye-research.org ; Tel.: +49-6131-17-3328
                Article
                jcm-08-01222
                10.3390/jcm8081222
                6723090
                31443184
                f9097434-c2dc-44f6-bf1b-0c5f2efeef31
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

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