Domain swapping is a wide spread phenomenon which involves the association between two or more protein subunits such that intra-molecular interactions between domains in each subunit are replaced by equivalent inter-molecular interactions between the same domains in different subunits. This thesis is devoted to the analysis of the factors that drive proteins to undergo such association modes. The specific system analyzed is the monomer to swapped dimer formation of the B1 domain of the immunoglobulin G binding protein (GB1). The formation of this dimer was shown to be fostered by 4 amino acid substitutions (L5V, F30V, Y33F, A34F) (Byeon et al., 2003). In this work, computational protein design and molecular dynamics simulations, both with detailed atomic models, were used to gain insight into how these 4 mutations may promote the domain swapping reaction. The stability of the wt and quadruple mutant GB1 monomers was assessed using the software DESIGNER, a fully automatic procedure that selects amino acid sequences likely to stabilize a given backbone structure (Wernisch et al., 2000). Results suggest that 3 of the mutations (L5V, F30V, A34F) have a destabilizing effect. The first mutation (L5V) forms destabilizing interactions with surrounding residues, while the second (F30V) is engaged in unfavorable interactions with the protein backbone, consequently causing local strain. Although the A34F substitution itself is found to contribute favorably to the stability of the monomer, this is achieved only at the expense of forcing the wild type W43 into a highly strained conformation concomitant with the formation of unfavorable interactions with both W43 and V54. Finally, we also provide evidence that A34F mutation stabilizes the swapped dimer structure. Although we were unable to perform detailed protein design calculations on the dimer, due to the lower accuracy of the model, inspection of its 3D structure reveals that the 34F side chains pack against one another in the core of the swapped structure, thereby forming extensive non-native interactions that have no counterparts in the individual monomers. Their replacement by the much smaller Ala residue is suggested to be significantly destabilizing by creating a large internal cavity, a phenomenon, well known to be destabilizing in other proteins. Our analysis hence proposes that the A34F mutation plays a dual role, that of destabilizing the GB1 monomer structure while stabilizing the swapped dimer conformation. In addition to the above study, molecular dynamics simulations of the wild type and modeled quadruple mutant GB1 structures were carried out at room and elevated temperatures (450 K) in order to sample the conformational landscape of the protein near its native monomeric state, and to characterize the deformations that occur during early unfolding. This part of the study was aimed at investigating the influence of the amino acid sequence on the conformational properties of the GB1 monomer and the possible link between these properties and the swapping process. Analysis of the room temperature simulations indicates that the mutant GB1 monomer fluctuates more than its wild type counter part. In addition, we find that the C-terminal beta-hairpin is pushed away from the remainder of the structure, in agreement with the fact that this hairpin is the structural element that is exchanged upon domain swapping. The simulations at 450 K reveal that the mutant protein unfolds more readily than the wt, in agreement with its decreased stability. Also, among the regions that unfold early is the alpha-helix C-terminus, where 2 out of the 4 mutations reside. NMR experiments by our collaborators have shown this region to display increased flexibility in the monomeric state of the quadruple mutant. Our atomic scale investigation has thus provided insights into how sequence modifications can foster domain swapping of GB1. Our findings indicate that the role of the amino acid substitutions is to decrease the stability of individual monomers while at the same time increase the stability of the swapped dimer, through the formation of non-native interactions. Both roles cooperate to foster swapping.

Quenched Molecular Dynamics (QMD) used to explore molecular conformations was developed to operate in Insight II platform for two simulation engines: CHARMm and Discover. Two scripts and procedures were written for molecular minimization, dynamics, minimization of each of several hundred conformers, and cut off. Experience with Insight II/Discover versus Quanta/CHARMm, and between Insight II/CHARMm versus Quanta/CHARMm has taught that the forcefield is the key factor in QMD studies. Protein A has been used for the purification of commercial antibodies, but it is expensive. Seven peptidomimetics of protein A were designed based on the hot-spots located at the helix-loop-helix region of protein A, and synthesized via solid phase using the Fmoc approach. These peptidomimetics were characterized by MS and NMR. The conformations of four peptidomimetics were studied by NMR and CD in water/hexafluoroisopropanol (pH 4). The CD and NMR data show that addition of hexafluoroisopropanol stabilizes their a-helical conformations. The structures of these peptidomimetics in solution were generated with Quanta/CHARMm using NMR data as limits for the QMD technique. Protein G has also been used to purify antibodies, but it is expensive too. A number of protein G mimics were designed as trivalent molecules. An efficient preparation of trivalent molecules having a useful primary amine arm has been developed through solid phase synthesis. The cheap, commercially available poly(propylene imine) dendrimers were used as scaffolds which allow multimerization of functionalized compounds. A small library of trivalent compounds were synthesized using this approach. A portion of compounds in this library were tested by Amersham Biosciences. The seven amino acid modified DAB-Am-4 exhibits strong binding to the IgG/Fab, and is a potential ligand for IgG purification. The interactions between neurotrophins (ie NGF and NT-3) and their receptors are typical drug targets. Fourteen second-generation peptidomimetics showing NGF-like or NT3-like activities in a preliminary bioassay, were resynthesized and tested again. Preliminary and retested data were compared. To access a direct binding assay, five fluorescently labeled peptidomimetics 41a-e were synthesized for a fluorescence activated cell sorting (FACScan) assay. Six monomeric precursors 42 and 43 were prepared on large scales for the library of bivalent turn analogs

Quenched Molecular Dynamics (QMD) used to explore molecular conformations was developed to operate in Insight II platform for two simulation engines: CHARMm and Discover. Two scripts and procedures were written for molecular minimization, dynamics, minimization of each of several hundred conformers, and cut off. Experience with Insight II/Discover versus Quanta/CHARMm, and between Insight II/CHARMm versus Quanta/CHARMm has taught that the forcefield is the key factor in QMD studies. Protein A has been used for the purification of commercial antibodies, but it is expensive. Seven peptidomimetics of protein A were designed based on the hot-spots located at the helix-loop-helix region of protein A, and synthesized via solid phase using the Fmoc approach. These peptidomimetics were characterized by MS and NMR. The conformations of four peptidomimetics were studied by NMR and CD in water/hexafluoroisopropanol (pH 4). The CD and NMR data show that addition of hexafluoroisopropanol stabilizes their a-helical conformations. The structures of these peptidomimetics in solution were generated with Quanta/CHARMm using NMR data as limits for the QMD technique. Protein G has also been used to purify antibodies, but it is expensive too. A number of protein G mimics were designed as trivalent molecules. An efficient preparation of trivalent molecules having a useful primary amine arm has been developed through solid phase synthesis. The cheap, commercially available poly(propylene imine) dendrimers were used as scaffolds which allow multimerization of functionalized compounds. A small library of trivalent compounds were synthesized using this approach. A portion of compounds in this library were tested by Amersham Biosciences. The seven amino acid modified DAB-Am-4 exhibits strong binding to the IgG/Fab, and is a potential ligand for IgG purification. The interactions between neurotrophins (ie NGF and NT-3) and their receptors are typical drug targets. Fourteen second-generation peptidomimetics showing NGF-like or NT3-like activities in a preliminary bioassay, were resynthesized and tested again. Preliminary and retested data were compared. To access a direct binding assay, five fluorescently labeled peptidomimetics 41a-e were synthesized for a fluorescence activated cell sorting (FACScan) assay. Six monomeric precursors 42 and 43 were prepared on large scales for the library of bivalent turn analogs

No description available.

Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment. / <p>QC 20160510</p>

Hypothalamic gonadotropin-releasing hormone (GnRH) (GnRH I) is the central regulator of the mammalian reproductive system. Most vertebrates studied also possess a second form of GnRH, GnRH II. GnRH I acts on its cognate G proteincoupled receptor (GPCR) on pituitary gonadotropes and activates Gq/11-mediated signalling pathways to stimulate the biosynthesis and the release of luteinising hormone (LH) and follicle-stimulating hormone (FSH). Both GnRHs have also been suggested to inhibit cellular proliferation, an action which has largely been proposed to be mediated by the coupling of the receptor to Gi/o. However, the range of G proteins activated by the GnRH receptor and the signalling cascades involved in inducing antiproliferation remain controversial. To delineate the G protein coupling selectivity of the mammalian GnRH receptor and to identify the signalling pathways involved in GnRH I-mediated cell growth inhibition, I examined the ability of the receptor to interact with Gq/11, Gi/o and Gs in Gαq/11 knockout MEF cells. My results indicate that the receptor is unable to interact with Gi/o but can signal through Gq/11. Additionally, my data do not support the suggestion of GnRH receptor-Gs interaction. Furthermore, I show that the GnRH Iinduced inhibition of cell growth is dependent on Gq/11, src and extracellular signal regulated kinase (ERK) but is independent of the activity of protein kinase C (PKC), Ca2+, jun-N-terminal kinase (JNK) or P38. Based on these findings and previous research within our group, I propose a mechanism whereby GnRH I may induce antiproliferation. Previous studies from our laboratory suggest that the GnRH receptor can adopt distinct active conformations in response to the binding of GnRH I and GnRH II. These data thus account for our hypothesis of ligand-induced selective signalling (LiSS). Given my previous results, I examined the ability of the GnRH receptor to couple to G12/13. My work indicates that the receptor can directly activate G12/13 and the downstream signalling cascades associated with this G protein family. Indeed, I provide evidence, in several cellular backgrounds, to suggest that GnRH receptor- G12/13-mediated signalling is involved in the regulation of GnRH-induced MAPK activity, SRE-driven gene transcription and cytoskeletal reorganisation. Furthermore, I propose a role for these G proteins in the transcriptional regulation of LHβ and FSHβ. Finally, I confirm previous results from our laboratory indicating that the GnRH receptor may interact with src Tyr kinase and show that GnRH I but not GnRH II may, independently of Gq/11, stimulate the Tyr phosphorylation and thus the activation of this protein. I propose that this differential signalling accounts for the distinct effects of GnRH I and GnRH II on cellular morphology and SREpromoted transcriptional activity. The research presented within this thesis provides evidence to refute published conclusions based on largely circumstantial experimental data, describes novel GnRH receptor signalling pathways and offers support for the concept of LiSS. It may assist in the development of new therapeutic compounds which selectively target one GnRH-mediated signalling pathway while bypassing others.

In protein engineering, rational design and selection from combinatorial libraries are methods used to develop proteins with new or improved features. A very important protein for the biological sciences is the antibody that is used as a detecting agent in numerous laboratory assays. Antibodies used for these purposes are often ”man-made”, by immunising animals with the desired target, or by selections from combinatorial libraries. Naturally, antibodies are part of the immune defence protecting us from foreign attacks from e.g. bacteria or viruses. Some bacteria have evolved surface proteins that can bind to proteins abundant in the blood, like antibodies and serum albumin. By doing so, the bacteria can cover themselves in the host’s own proteins and through that evade being detected by the immune system. Two such proteins are Protein A from Staphylococcus aureus and Protein G from group C and G Streptococci. Both these proteins contain domains that bind to antibodies, one of which is denoted C2 (from Protein G) and another B (from Protein A). The B domain have been further engineered to the Z domain. In this thesis protein engineering has been used to develop variants of the C2 and Z domains for site-specific labelling of antibodies and for antibody purification with mild elution. By taking advantage of the domains’ inherent affinity for antibodies, engineering and design of certain amino acids or protein motifs of the domains have resulted in proteins with new properties. A photo crosslinking amino acid, p-benzoylphenylalanine, have been introduced at different positions to the C2 domain, rendering three new protein domains that can be used for site-specific labelling of antibodies at the Fc or Fab fragment. These domains were used for labelling antibodies with lanthanides and used for detection in a multiplex immunoassay. Moreover, a library of calcium-binding loops was grafted onto the Z domain and used for selection of a domain that binds antibodies in a calcium dependent manner. This engineered protein domain can be used for the purification of antibodies using milder elution conditions, by calcium removal, as compared to traditional antibody purification.

Pulmonale Surfactant-Proteine sind seit vielen Jahrzehnten bekannt und wurden erstmals in der Lunge beschrieben, wo sie wichtige Funktionen wie die Reduktion der Oberflächenspannung der Alveolarflüssigkeit erfüllen. Aufgrund dieser Eigenschaft stellen diese Proteine Eckpfeiler in der Therapie des Atemnotsyndroms von Frühgeborenen dar, seitdem sie synthetisch produziert werden können. Während der letzten Jahre wuchs das Interesse an zerebralen Surfactant-Proteinen, die rheologische Eigenschaften von Grenzmembranen beeinflussen sowie immunmodulatorische Aufgaben erfüllen und in Verbindung mit Entzündungen des ZNS sowie hydrozephalen Zuständen stehen. Das Surfactant-Protein-G (SP-G) wurde ebenfalls zunächst in der Lunge nachgewiesen, später aber auch im Gehirn. Dort wurde es z.B. entlang der Blut-Hirn-Schranke detektiert und zeigte veränderte Proteinlevel bei intrazerebralen Blutungen, Infektionen oder dem posthämorrhagischen Hydrozephalus. Weiterhin wird eine mögliche Rolle des SP-G für das glymphatische System diskutiert. Es galt lange Zeit als sicher, dass dem ZNS ein Lymphgefäßsystem fehlt und Schadstoffe über den Liquor cerebrospinalis in die venösen Duralsinusse geleitet werden. Seit einigen Jahren ändert sich jedoch das Bild von der „Abfallentsorgung“ im Gehirn, wobei die Astroglia eine zentrale Rolle zu spielen scheint. Mit ihren Endfüßen umgeben diese Zellen die intrazerebralen Arterien und erzeugen über polarisierte Aquaporin 4 (AQP4) -Kanäle einen durch arterielle Pulsation ausgelösten, gerichteten Einstrom von Flüssigkeit aus dem periarteriellen Raum in das Hirnparenchym. Nachdem sie das Parenchym durchflossen haben, werden die transportierten Flüssigkeiten über perivenöse und perineuronale Räume sowie über Lymphgefäße innerhalb der Duralsinusse zu den extrakraniellen Lymphknoten geleitet. Dieses System wird als Glia-lymphatisches, kurz glymphatisches, System bezeichnet. Eine Störung des Flüssigkeitsstroms führt auch zu einer gestörten Entsorgung von schädlichen Metaboliten, darunter β-Amyloid (Aβ). Hauptsächlich aus Aβ bestehende senile Plaques und Neurofibrillen aus hyperphosphoryliertem Tau sind die histopathologischen Hauptkennzeichen der Alzheimerschen Erkrankung (AD), der mit Abstand häufigsten Demenzform. Weltweit sind etwa 50 Millionen Menschen von der AD betroffen, was zu enormen familiären, gesellschaftlichen und ökonomischen Belastungen führt. Steigendes Alter ist der Hauptrisikofaktor für das Auftreten der AD, sodass diese Belastungen insbesondere in der alternden deutschen Bevölkerung noch weiter zunehmen werden. Hieraus erklärt sich das enorme Forschungsinteresse an dieser Krankheit. Dabei ermöglichen Tiermodelle für einzelne Aspekte der AD Untersuchungen ihrer Pathogenese sowie die Entwicklung möglicher Therapie- und Behandlungsansätze. Die vorliegende Studie basiert auf dem etablierten 3xTg-Mausmodell, das Alzheimer-artige Veränderungen und kognitive Einschränkungen aufweist, sowie altersgleichen Wildtyp-Mäusen, die als Vergleichsgruppe dienten. Hauptziele der durchgeführten Untersuchungen waren immunhistochemische Analysen der Lokalisation von SP-G im Vorderhirn von Mäusen, die Lagebeziehungen von SP-G-Immunreaktivität (Ir) zu Komponenten der Neurovaskulären Einheit (NVU) und zu AD-Läsionen in alten 3xTg-Mäusen sowie biochemische Analysen der Proteinlevel von SP-G im Gehirn von Versuchstieren unterschiedlichen Alters und Genotyps. Mittels Fluoreszenz-Dreifachmarkierungen wurden zunächst die Lagebeziehungen zwischen SP-G-Ir und neuronalen, glialen sowie vaskulären Komponenten der NVU detektiert. Der Nachweis von Nervenzellen gelang durch indirekte Immunfluoreszenz mit Antikörpern gegen neuronale Nuklei (NeuN), Mikroglia wurden mit anti-Iba (Ionisiertes Kalium-bindendes Adapter-Molekül 1) visualisiert, während Astrozyten durch die Marker GFAP (Saures Gliafaserprotein) und S100β sowie astrozytäre Endfüße durch AQP4-Ir sichtbar wurden. Biotinylierte Lektine aus der Kartoffel (Solanum tuberosum Lektin, STL) und der Tomate (Lycopersicon esculentum Agglutinin, LEA) dienten zusammen mit fluorochromierten Streptavidin-Derivaten der simultanen Detektion von Gefäßen. Die vorliegende Studie zeigt erstmals SP-G-immunreaktive Somata und Zellfortsätze von Neuronen z.B. in der Habenula, im Hypothalamus und im Infundibulum. SP-G-Ir in sensorischen Fortsätzen bipolarer Interneurone, die in Kontakt mit dem Liquor cerebrospinalis stehen, ist ein Hinweis auf eine mögliche Rolle des Proteins bei Kontrolle und Regulation von Liquorzusammensetzung und -fließgeschwindigkeit. Zusätzlich vorliegende vorläufige Befunde zu SP-G-Ir in mit dem Plexus choroideus assoziierten Iba-positiven Kolmer-Zellen bleiben noch zu validieren. Ein weiteres wichtiges Ergebnis der vorliegenden Arbeit war der Nachweis von SP-G-Ir in dotartigen, häufig geclustert auftretenden Ablagerungen vor allem im Hippokampus, aber auch im piriformen Kortex. Die SP-G-Dots zeigten sich in 3xTg-Mäusen mit altersabhängigen Aβ-Plaques und hyperphosphoryliertem Tau stets räumlich getrennt von diesen Läsionen. Die geclustert auftretenden Dots waren auch immunpositiv für Reelin und ähnelten im Erscheinungsbild den in früheren Studien detektierten Ablagerungen z.B. von Laminin-bindendem Protein und SP-C. Ein Hauptgegenstand der vorliegenden Studie war die computergestützte Semiquantifizierung von SP-G-immunreaktiven hippokampalen Dots in insgesamt 55 3xTg- und Wildtyp-Mäusen im Alter von 3, 6, 12, 16 und 24 Monaten. Dabei ergab sich unabhängig vom Genotyp eine starke und signifikante Zunahme der SP-G-immunpositiven Ablagerungen im Altersgang der untersuchten Tiere. Außerdem zeigten 3xTg-Mäuse einen signifikant erhöhten hippokampalen Anteil der dotartigen Ablagerungen im Vergleich mit den altersentsprechen Wildtyp-Mäusen. Abschließender Schwerpunkt waren biochemische Analysen zur Quantifizierung des SP-G-und Reelin-Gehalts in der Gesamtproteinfraktion sowie im intra- und extrazellulären Kompartiment des ZNS von 3xTg- und Wildtyp-Mäusen. Die durchgeführten Enzymimmunassays (ELISAs) umfassten Hirnproben von 67 Tieren im Alter zwischen 3 und 24 Monaten. Dabei korrelierte das zunehmende Alter der 3xTg-Mäuse mit erhöhten SP-G-Konzentrationen, während sich bei Wildtyp-Tieren keine signifikante Veränderung im Altersgang nachweisen ließ. Zudem zeigten die transgenen Tiere höhere SP-G-Level als altersgleiche Wildtypen. Darüber hinaus waren auch die Reelin-Level aller Altersgruppen in 3xTg-Mäusen höher als in Wildtyp-Mäusen. Eine Korrelation zwischen zunehmendem Alter und Reelingehalt konnte für 3xTg-Mäuse nachgewiesen werden, wogegen sich diese Korrelation in Wildtyp-Tieren invers zeigte. Die hier vorgelegten Ergebnisse deuten auch auf eine mögliche Rolle von SP-G bei der Pathogenese der AD hin. Dennoch bildet diese Arbeit nur einen Ausgangspunkt für zukünftige Studien von Funktionen des SP-G innerhalb des glymphatischen Systems und für die zerebrale Immunabwehr. Zusätzliche elektronenmikroskopische Analysen von SP-G in Gehirnen weiterer Säugetier-Spezies sowie in autoptischen humanen Proben bleiben wünschenswert.

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively preconcentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, an on-line protein G monolithic preconcentrator CE system for enrichment and separation of proteins was developed. This system proved effective for on-line sample extraction, clean-up, preconcentration, and CE of IgG in human serum. IgG from diluted (500 and 65,000 times) human serum samples was successfully analyzed using this system. The approach can be applied to the on-line preconcentration and analysis of any protein for which an antibody is available. The desire to separate all proteins present in human tissues, cells and biological fluids has challenged the separation research community for many years. The difficulty of this task resides in the complexity of the sample. Blood serum, for instance, may express up to 10,000 proteins with an estimated dynamic range of 9 orders of magnitude. Additionally, most of these proteins are present at very low concentrations (ng/mL). Identification and quantification of low abundance proteins is hindered by the presence of high abundance proteins, such as human serum albumin (HSA) and immunoglobulins (IgG). Therefore, in most cases, removal of the high abundance proteins or enrichment of low abundance proteins is necessary prior to the analysis of low abundance proteins. To address this, a coupled affinity-hydropobic monolithic column for the simultaneous removal of IgG, preconcentration of low abundance proteins, and separation by capillary zone electrophoresis was designed. The system proved to be very reproducible. The run-to-run %RSD values for migration time and peak area were less than 5%, which is typical of CE. Finally, a new method was developed to prepare monoliths with anion exchange functionality. Polymer monoliths were prepared by in situ polymerization of methacrylate monomers. The monoliths were coated with a water soluble polymer and used for the analysis of proteins. Using this approach, a model monolith was prepared. Subsequent coating yielded a monolith with quaternary ammonium groups on the surface, which was confirmed by strong anodic electroosmotic flow. Analysis of standard proteins by ion exchange LC and CEC was demonstrated. This simple and rapid method for surface modification opened new avenues for the preparation of monoliths with a broad range of functionalities.

A ativação excessiva dos receptores NMDA tem sido descrita no disparo da morte neuronal que ocorre em doenças, como o glaucoma. É possível que a combinação de subunidades (NR2A-D) possa ativar vias de sinalização intracelulares que resultam na morte ou sobrevivência. Nosso objetivo foi investigar o envolvimento de subunidades NR2 e Rac1, membro da família Rho GTPase, na morte de neurônios da retina. A morte induzida por glutamato in vitro foi reduzida após a inibição de Rac1 e bloqueio de NR2B, mas não das subunidades NR2C/D. Resultados semelhantes foram obtidos in vivo após injeção intravítrea NMDA, e a detecção de Rac1 ativo, principalmente, nos processos de glia de Müller foi inibida pelo bloqueio NR2B. Além disso, a produção de TNF-<font face=\"Symbol\">&#945; após a injeção de NMDA foi reduzida pelo bloqueio de NR2B e Rac1. Assim, nossos resultados sugerem que a excitotoxicidade via receptores NR2B/NMDA ativa Rac1 em células da glia de Müller, que por sua vez controla a produção de TNF-<font face=\"Symbol\">&#945 possível responsável pela morte de células ganglionares da retina. / Overactivation of NMDA receptors has been described to trigger neuronal death that occurs in diseases such as glaucoma. It is possible that the combination of subunits (NR2A-D) activate intracellular signaling pathways that result in death or survival. Our aim was to investigate the involvement of NR2 subunits and Rac1, a member of Rho GTPase family, in retinal neuronal death. Glutamate-induced neuronal death in vitro was reduced after Rac1 inhibition and by NR2B blocking, but not NR2C/D subunits. Similar results were obtained in vivo after NMDA intravitreous injection, although active Rac1 was mainly detected in Müller glia processes, and it was inhibited by NR2B blockade. In addition, TNF-<font face=\"Symbol\">&#945 level after NMDA injection were reduced by NR2B blocking and Rac1. Thus, our results suggest that excitotoxicity via NR2B/NMDA receptors activate Rac1 in Müller glia cells, which in turn controls the TNF-<font face=\"Symbol\">&#945 production that triggers retinal ganglion cell death.