ETX1 is over-expressed in the glaucomatous trabecular meshwork

To determine whether exon-trapped X chromosome clone 1 (ETX1) is overexpressed in the trabecular meshwork (TM) of glaucomatous human eyes compared to controls. Immunohistochemical, western blot, and enzyme-linked immunosorbent assay analysis were used with human tissues and TM protein extracts. Reverse transcription-PCR was performed on isolated mRNA-derived cDNA preparations. Elevated expression levels of ETX1 were detected in glaucomatous compared to control TM tissue. This corroborates previous detection of ETX1 in glaucomatous TM by proteomic analysis. ETX1 mRNA is present in TM tissue, suggesting ETX1 protein is locally produced within TM cells. This is the first report demonstrating overexpression of ETX1 in glaucomatous TM. ETX1 expression may regulate TM protein interactions involved in cell adhesion, and its aberrant overexpression may be part of the pathophysiological pathway in the development of glaucoma

Activación de astrocitos por deiminación post-traduccional

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2016.Una relación en el nivel elevado y aberrante en la deiminación post traduccional ha sido demostrado en estudios relacionados a la esclerosis múltiple (MS) (Bates et al, 2002; Kim et al, 2003) y más recientemente en el nervio óptico de pacientes con glaucoma con alta presión intraocular (Bhattacharya et al., 2006) y en pacientes con glaucoma con presión intraocular normal (Cafaro et al, 2010). La esclerosis múltiple es vista como una enfermedad autoinmune y algunos componentes de la autoinmunidad también han sido relacionados con el glaucoma (Tezel & Wax, 2007). Nosotros intentamos demostrar que el nivel de expresión de la enzima deiminasa de la peptidyl arginina 2 (PAD2) es más elevado en los astrocitos a diferencia de otras células como respuesta a una variedad de parámetros fisicoquímicos. Nuestro trabajo preliminar demuestra que la elevación de la presión ambiental es uno de esos parámetros (Algeciras et al, 2008). También hemos demostrado que la elevación en el nivel de PAD2 está asociada con una elevación en el nivel de parámetros bioquímicos relacionados con la activación de astrocitos (Govindarajan et al, 2009). En mi tesis yo evaluaré diferentes estrategias para intentar elevar los niveles de PAD2 y consecuentemente la deiminación, así como intentaré también llegar a un estimado cuantitativo de activación bioquímica. Finalmente, utilizaré anticuerpos y diferentes técnicas para tratar de demostrar que los astrocitos pueden ser capaces de expresar moléculas importantes en la presentación antigénica y potencialmente estar capacitados, bajos condiciones de activación, para actuar como células presentadores de antígeno y activar a los linfocitos T. Nuestra 3 hipótesis es que la elevación en la deiminación post traduccional en el nervio óptico de pacientes con glaucoma o en el tejido cerebral de pacientes con esclerosis múltiple puede desencadenar una cascada lenta y progresiva: la modificación de las proteínas de la mielina por la deiminación post traduccional genera productos auto catalíticos que activan a los astrocitos. Cuando los astrocitos son activados, pueden obtener habilidades de células presentadoras de antígenos y así iniciar una reacción con participación de diferentes componentes inmunológicos que contribuye a la muerte de las neuronas en ciertas enfermedades neurodegenerativas.Algeciras, Mabel Enriquez. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Bhattacharya, Sanjoy K. Universidad de Miami. Departamento de Oftalmología. Bascom Palmer Eye. Institute; Estados Unidos de Norteamérica.Serra, Horacio Marcelo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Iribarren, Pablo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Quiroga, Santiago. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.Radrizzani, Martín. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Laboratorio de Neurología y Citogenética Molecular; Argentina

Lipidomic mass spectrometry and its application in neuroscience

Central and peripheral nervous systems are lipid rich tissues. Lipids, in the context of lipid-protein complexes, surround neurons and provide electrical insulation for transmission of signals allowing neurons to remain embedded within a conducting environment. Lipids play a key role in vesicle formation and fusion in synapses. They provide means of rapid signaling, cell motility and migration for astrocytes and other cell types that surround and play supporting roles neurons. Unlike many other signaling molecules, lipids are capable of multiple signaling events based on the different fragments generated from a single precursor during each event. Lipidomics, until recently suffered from two major disadvantages: (1) level of expertise required an overwhelming amount of chemical detail to correctly identify a vast number of different lipids which could be close in their chemical reactivity; and (2) high amount of purified compounds needed by analytical techniques to determine their structures. Advances in mass spectrometry have enabled overcoming these two limitations. Mass spectrometry offers a great degree of simplicity in identification and quantification of lipids directly extracted from complex biological mixtures. Mass spectrometers can be regarded to as mass analyzers. There are those that separate and analyze the product ion fragments in space (spatial) and those which separate product ions in time in the same space (temporal). Databases and standardized instrument parameters have further aided the capabilities of the spatial instruments while recent advances in bioinformatics have made the identification and quantification possible using temporal instruments

Lipidomic mass spectrometry and its application in neuroscience MINIREVIEWS

Central and peripheral nervous systems are lipid rich tissues. Lipids, in the context of lipid-protein complexes, surround neurons and provide electrical insulation for transmission of signals allowing neurons to remain embedded within a conducting environment. Lipids play a key role in vesicle formation and fusion in synapses. They provide means of rapid signaling, cell motility and migration for astrocytes and other cell types that surround and play supporting roles neurons. Unlike many other signaling molecules, lipids are capable of multiple signaling events based on the different fragments generated from a single precursor during each event. Lipidomics, until recently suffered from two major disadvantages: (1) level of expertise required an overwhelming amount of chemical detail to correctly identify a vast number of different lipids which could be close in their chemical reactivity; and (2) high amount of purified compounds needed by analytical techniques to determine their structures. Advances in mass spectrometry have enabled overcoming these two limitations. Mass spectrometry offers a great degree of simplicity in identification and quantification of lipids directly extracted from complex biological mixtures. Mass spectrometer

Mechanical stretching elevates peptidyl arginine deiminase 2 expression in astrocytes

To determine whether the mechanical stretching renders modulation of the peptidyl arginine deiminase 2 (PAD2) expression in cultured astrocytes. Isolated rat brain astrocytes were subjected to mechanical stretching using a glass bead set-up and polyethylene set-up with or without immobilization. Activity assays and ELISA were performed to detect PAD2 expression. Protein deimination levels in the cells were measured using an anti-citrulline ELISA. PAD2 expression in cells subjected to mechanical stretching was compared with controls. Astrocytes were also subjected to pressure treatment and compared to control cells for PAD2 expression and deimination levels. Astrocytes subjected to mechanical stretching with or without immobilization showed elevated PAD2 expression. Pressure treatment of astrocytes (25-100 mmHg) also resulted in elevated PAD2 expression and deimination. These results suggest mechanical stretching induced PAD2 expression and consequent protein deimination

Protein Deimination in Aging and Age-Related Diseases with Ocular Manifestations

Deimination refers to the conversion of protein-bound arginines into citrulline. It has been established as a posttranslational modification due to the lack of any known tRNA carrier for citrulline, as well as the presence of deiminases that are capable of catalyzing this modification in vitro. There is no known enzyme that can revert protein-bound citrulline into arginine, rendering it a relatively long-term modification. Elevated deimination has been found in neuronal tissues in a number of neurodegenerative diseases including multiple sclerosis and glaucoma. Observations in the retina, a tissue where the retinal ganglion cell layer lacks a substantial presence of astroglial cells, demonstrated that elevated and reduced deimination occurs simultaneously in astroglial cells and neurons, respectively. Such opposite effects are expected to complicate therapeutic strategies, necessitating cell-specific delivery systems for perturbation of deiminases that catalyze deimination in neuronal tissues. In this review, we will briefly discuss the occurrence of deimination with normal aging, the importance of deimination in diseases, and the effect of deimination on mRNA transport in neuronal tissue. Elevated deimination induces proteolysis via modification of protein structures, while reduced deimination affects protein synthesis and the outgrowth of dendrites in neurons

Chemical Modification and Mass Spectrometric Approaches for Detection of Brain Protein Deimination

The posttranslational modification of deimination refers to conversion of protein-bound arginine into citrulline. It is frequently detected by monoxime treatment (acid phase 2,3-butanedione and antipyrine reaction) resulting in an adduct formation. We present evidence that deiminated proteins are prone to aggregate formation upon prolonged monoxime exposure. Moreover, the efficiency of adduct formation is nonlinear and dramatically reduced with increasing polypeptide complexity [complete with smaller but progressively decreasing with higher (poly)peptides]. This nonlinearity results in vastly noncomparable detection among different methods such as immunohistochemistry, Western blot, and mass spectrometry. Mass spectrometric detection, based on mass addition of +238 on citrulline moiety with monoxime and +1 change due to deimination without monoxime treatment, corroborates serious limitations in monoxime adduct formation on proteins. Methodological limitations may also interfere with the identification of deiminated proteins as well as their modification sites and, as a consequence, the understanding of the biological role of deimination. We present here methods that alter the sequence of digestion and the combination of chromatographic techniques that collectively reduce complexity and help capture the deiminated peptides. Thin-layer chromatographic methods, together with different enzymatic digestions, can also be potentially routinely used for detection of changes in deimination sites within a given protein in different states of a cell or tissue

Deimination in Ocular Tissues: Present and Future

Deimination refers to the posttranslational conversion of protein-bound arginines into protein-bound citrullines, catalyzed by peptidyl arginine deiminase (PAD) enzymes (Vossenaar et al. 2003). Lack of a carrier tRNA for citrulline has established deimination as a posttranslational modification. Deimination and citrullination are interchangeably used for conversion of arginine into citrulline. To distinguish free arginine citrullination from protein-bound citrullination, we prefer referring to the latter as deimination (Bhattacharya 2009). Throughout this chapter we use deimination to refer to the conversion of protein-bound arginine modification into citrulline