Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

The molecular role of the common gamma c subunit in signal transduction reveals functional asymmetry within multimeric cytokine receptor complexes.

The specific signal transduction function of the gamma c subunit in the interleukin (IL) 2, IL-4, IL-7, IL-9, and IL-15 receptor complexes remains undefined. The present structure-function analyses demonstrated that the entire cytoplasmic tail of gamma c could be functionally replaced in the IL-2 receptor (IL-2R) signaling complex by a severely truncated erythropoietin receptor cytoplasmic domain lacking tyrosine residues. Heterodimerization of IL-2R beta with either gamma c or the truncated erythropoietin receptor chain led to an array of specific signals normally derived from the native IL-2R despite the substitution of Janus kinase JAK2 for JAK3 in the receptor complex. These findings thus suggest a model in which the gamma c subunit serves as a common and generic "trigger" chain by providing a nonspecific Janus kinase for signaling program initiation, while signal specificity is determined by the unique "driver" subunit in each of the gamma c- containing receptor complexes. Furthermore, these results may have important functional implications for the asymmetric design of many cytokine receptor complexes and the evolutionary design of receptor subfamilies that share common trigger or driver subunits.

Identification of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a combinatorial chemical library.

Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, and the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN.

Structural and functional analysis of pp70S6k.

The pp70/85-kDa S6 kinases, collectively referred to as pp70S6k, are thought to participate in transit through the G1 phase of the cell cycle. pp70S6k regulates the phosphorylation of the 40S ribosomal protein S6 and the transcription factor CREM tau. pp70S6k is regulated by serine/threonine phosphorylation, and although 1-phosphatidylinositol 3-kinase and phospholipase C have been implicated as upstream regulators, the mechanism of activation and identity of the upstream pp70S6k kinases remain unknown. To improve our understanding of how this mitogen-stimulated protein kinase is regulated by growth factors and the immunosuppressant rapamycin, we have initiated a structure/function analysis of pp70S6k. Our results indicate that both the N and C termini participate in the complex regulation of pp70S6k activity.

Expression studies of catalytic antibodies.

We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived from a number of catalytic antibodies. Expression yields of eight hybridoma- and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high density fermentations. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies.

Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor.

Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an alpha-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist [D-Phe12,Nle21,38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Glu-Xaa-Xaa-Lys scaffold. We have found astressin [cyclo(30-33)[D-Phe12,Nle21,38,Glu30,Lys33]r/ hCRF(12-41)] to be approximately 30 times more potent than [D-Phe12,Nle21,38]r/hCRF-(12-41), our present standard, and 300 times more potent than the corresponding linear analog in an in vitro pituitary cell culture assay. Astressin has low affinity for the CRF binding protein and high affinity (Ki = 2 nM) for the cloned pituitary receptor. Radioiodinated [D-125I-Tyr12]astressin was found to be a reliable ligand for binding assays. In vivo, astressin is significantly more potent than any previously tested antagonist in reducing hypophyseal corticotropin (ACTH) secretion in stressed or adrenalectomized rats. The cyclo(30-33)[Ac-Pro4,D-Phe12,Nle21,38,Glu30,Lys33++ +]r/hCRF-(4-41) agonist and its linear analog are nearly equipotent, while the antagonist astressin and its linear form vary greatly in their potencies. This suggests that the lactam cyclization reinstates a structural constraint in the antagonists that is normally induced by the N terminus of the agonist.

Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts.

The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.

High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N omega-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription.

NGFI-A (also called Egr1, Zif268, or Krox24) and the closely related proteins Krox20, NGFI-C, and Egr3 are zinc-finger transcription factors encoded by immediate-early genes which are induced by a wide variety of extracellular stimuli. NGFI-A has been implicated in cell proliferation, macrophage differentiation, synaptic activation, and long-term potentiation, whereas Krox20 is critical for proper hindbrain segmentation and peripheral nerve myelination. In previous work, a structure/function analysis of NGFI-A revealed a 34-aa inhibitory domain that was hypothesized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid system, we have isolated a cDNA clone which encodes a protein that interacts with this inhibitory domain and inhibits the ability of NGFI-A to activate transcription. This NGFI-A-binding protein, NAB1, is a 570-aa nuclear protein that bears no obvious sequence homology to known proteins. NAB1 also represses Krox20 activity, but it does not influence Egr3 or NGFI-G, thus providing a mechanism for the differential regulation of this family of immediate-early transcription factors.

Residues in the pathway through a membrane transporter.

The structure of solute transporters is understood largely from analysis of their amino acid sequences, and more direct information is greatly needed. Here we report work that applies cysteine scanning mutagenesis to describe structure-function relations in UhpT, a bacterial membrane transporter. By using an impermeant SH-reactive agent to probe single-cysteine variants, we show that UhpT transmembrane segment 7 spans the membrane as an alpha-helix and that the central portion of this helix is exposed to both membrane surfaces, forming part of the translocation pathway through this transporter.

A paradigm for drug discovery employing encoded combinatorial libraries.

Very large combinatorial libraries of small molecules on solid supports can now be synthesized and each library element can be identified after synthesis by using chemical tags. These tag-encoded libraries are potentially useful in drug discovery, and, to test this utility directly, we have targeted carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) as a model. Two libraries consisting of a total of 7870 members were synthesized, and structure-activity relationships based on the structures predicted by the tags were derived. Subsequently, an active representative of each library was resynthesized (2-[N-(4-sulfamoylbenzoyl)-4'-aminocyclohexanespiro]-4-oxo-7 -hydroxy- 2,3-dihydrobenzopyran and [N-(4-sulfamoylbenzoyl)-L-leucyl]piperidine-3-carboxylic acid) and these compounds were shown to have nanomolar dissociation constants (15 and 4 nM, respectively). In addition, a focused sublibrary of 217 sulfamoylbenzamides was synthesized and revealed a clear, testable structure-activity relationship describing isozyme-selective carbonic anhydrase inhibitors.

Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Dalla catalisi all’inibizione della crescita da contatto: le molteplici funzioni di O-acetilserina sulfidrilasi batterica

CysK, uno degli isoenzimi di O-acetilserina sulfidrilasi (OASS) presenti in piante e batteri, è un enzima studiato da molto tempo ed il suo ruolo fisiologico nella sintesi della cisteina è stato ben definito. Recentemente sono state scoperte altre funzioni apparentemente non collegate alla sua funzione enzimatica (moonlighting). Una di queste è l’attivazione di una tossina ad attività tRNAsica, CdiA-CT, coinvolta nel sistema di inibizione della crescita da contatto (CDI) di ceppi patogeni di E. coli. In questo progetto abbiamo studiato il ruolo di CysK nel sistema CDI e la formazione di complessi con due differenti partner proteici: CdiA-CT e CysE (serina acetiltransferasi, l’enzima che catalizza la reazione precedente nella biosintesi della cisteina). I due complessi hanno le stesse caratteristiche spettrofluorimetriche e affinità molto simili, ma la cinetica di raggiungimento dell’equilibrio per il complesso tossina:CysK è più lenta che per il complesso CysE:CysK (cisteina sintasi). In entrambi i casi la formazione veloce di un complesso d’incontro è seguita da un riarrangiamento conformazionale che porta alla formazione di un complesso ad alta affinità. L’efficienza di formazione del complesso cisteina sintasi è circa 200 volte maggiore rispetto al complesso CysK:tossina. Una differenza importante, oltre alla cinetica di formazione dei complessi, è la stechiometria di legame. Infatti mentre CysE riesce a legare solo uno dei due siti attivi del dimero di CysK, nel complesso con CdiA-CT entrambi i siti attivi dell’enzima risultano essere occupati. Le cellule isogeniche esprimono un peptide inibitore della tossina (CdiI), e sono quindi resistenti all’azione tRNAsica. Tuttavia, siccome CdiI non altera la formazione del complesso CdiA-CT:CysK, CdiA-CT può esercitare comunque un ruolo nel metabolismo della cisteina e quindi nella fitness dei batteri isogenici, attraverso il legame e l'inibizione di CysK e la competizione con CysE. La via biosintetica della cisteina, un precursore di molecole riducenti, risulta essere molto importante per i batteri soprattutto in condizioni avverse come all’interno dei macrofagi nelle infezioni persistenti. Perciò questa via metabolica è di interesse per lo sviluppo di nuovi antibiotici, e in particolare le due isoforme dell’OASS negli enterobatteri, CysK e CysM, sono potenziali target per lo sviluppo di nuove molecole ad azione antibatterica. Partendo dall’analisi delle modalità di interazione con CysK del suo partner ed inibitore fisiologico, CysE, si è studiato dapprima l’interazione di pentapeptidi che mimassero la regione C-terminale di quest'ultimo, e in base ai dati ottenuti sono stati sviluppati piccoli ligandi sintetici. La struttura generale di questi composti è costituita da un gruppo acido ed un gruppo lipofilo, separati da un linker ciclopropanico che mantiene questi due gruppi in conformazione trans, ottimale per l’interazione col sito attivo dell’enzima. Sulla base di queste considerazioni, di docking in silico e di dati sperimentali ottenuti con la tecnica dell’STD-NMR e con saggi di binding spettrofluorimetrici, si è potuta realizzare una analisi di relazione struttura-attività che ha portato via via all’ottimizzazione dei ligandi. Il composto più affine che è stato finora ottenuto ha una costante di dissociazione nel range del nanomolare per entrambe le isoforme, ed è un ottimo punto di partenza per lo sviluppo di nuovi farmaci.

Antichagásicos potenciais: síntese e modelagem molecular de híbridos de hidrazonas e liberadores de óxido nítrico

A doença de Chagas é uma parasitose extremamente negligenciada, cujo agente etiológico é o protozoário Trypanosoma cruzi. Atualmente, 21 países da América Latina são considerados regiões endêmicas, onde 75-90 milhões de pessoas estão expostas à infecção, 6-7 milhões estão infectadas e mais de 41 mil novos casos surgem por ano. Entretanto, apenas os fármacos nifurtimox e benznidazol estão disponíveis no mercado. Estes, além da baixa eficácia na fase crônica da parasitose, apresentam diversos efeitos adversos, sendo que no Brasil apenas o benznidazol é utilizado. Este fato mostra a importância de se ampliar o número de fármacos disponíveis e propor quimioterapia mais eficaz para o tratamento da doença de Chagas. Como forma de contribuir para essa busca, este trabalho objetiva a síntese de compostos híbridos bioisostéricos N-acilidrazônicos e sulfonilidrazônicos, contendo grupo liberador de óxido nítrico, com potencial de interação com cisteíno-proteases parasitárias, tais como a cruzaína. Nestes derivados, os grupos liberadores de óxido nítrico utilizados foram os grupos furoxano (contendo substituinte metílico e fenílico) e éster nitrato. Propôs-se a variação de anéis aromáticos substituídos e não-substituídos, com o intuito de avaliar a possível relação estrutura-atividade (REA) desses análogos. Até o momento, somente os compostos da série N-acilidrazônica tiveram avaliação biológica realizada. Os valores de IC50 dos compostos na forma amastigota do parasita variaram entre >100 a 2,88 µM, sendo este último valor comparável ao fármaco de referência. A atividade inibitória frente à cruzaína foi de 25,2 µM a 2,2 µM. Já a liberação de óxido nítrico foi avaliada pelo método indireto de detecção de nitrato e os valores variaram entre 52,0 µM e 4.232,0 µM. Estes são bem inferiores ao composto padrão, além de não se identificar correlação direta entre a atividade biológica e a liberação de NO. Na sequência, os dois compostos mais ativos (6 e 14) foram submetidos a estudos de permeabilidade e de citotoxicidade. O composto 6 foi considerado o de maior permeabilidade segundo o Sistema de Classificação Biofarmacêutica (SCB) e todos os compostos apresentaram a taxa de fluxo menor que 2, indicando a ausência de mecanismo de efluxo. Na avaliação do potencial citotóxico desses compostos em células humanas, o derivado 6 apresentou índice de seletividade superior ao do benznidazol. Em estudos de modelagem molecular usando análise exploratória de dados (HCA e PCA), propriedades estéricas/geométricas e eletrônicas foram consideradas as mais relevantes para a atividade biológica. Além disso, estudos de docking mostraram que a posição do grupo nitro no anel aromático é importante para a interação com a cruzaína. Ademais o composto 6 não provocou mudanças significativas no ciclo celular e na fragmentação de DNA em células humanas, mostrando-se como líder promissor para futuros estudos in vivo. Atividade tripanomicida, citotoxicidade, potencial de liberação de NO e estudos de permeabilidade dos 23 derivados sulfonilidrazônicos e ésteres nitrato estão sendo avaliados.

Reverse engineering as a teaching tool: the corneal topographer

Póster presentado en EDULEARN12, International Conference on Education and New Learning Technologies, Barcelona, 2nd-4th July 2012.