SNAP-25 and synaptotagmin involvement in the final Ca(2+)-dependent triggering of neurotransmitter exocytosis.

In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca(2+)-signaled exocytosis is effectively blocked by antibodies and peptides to SNAP-25, a key constituent of the vesicle docking complex. In addition, Ca2+ inhibits the ability of synaptotagmin, a synaptic vesicle protein proposed as a calcium sensor and triggering device, to associate with this docking complex. These results support a model in which Ca(2+)-dependent triggering of neurotransmission at central synapses acts after ATP-dependent potentiation of the docking-fusion complex for membrane fusion.

Unraveling principles of lead discovery: from unfrustrated energy landscapes to novel molecular anchors.

The search for novel leads is a critical step in the drug discovery process. Computational approaches to identify new lead molecules have focused on discovering complete ligands by evaluating the binding affinity of a large number of candidates, a task of considerable complexity. A new computational method is introduced in this work based on the premise that the primary molecular recognition event in the protein binding site may be accomplished by small core fragments that serve as molecular anchors, providing a structurally stable platform that can be subsequently tailored into complete ligands. To fulfill its role, we show that an effective molecular anchor must meet both the thermodynamic requirement of relative energetic stability of a single binding mode and its consistent kinetic accessibility, which may be measured by the structural consensus of multiple docking simulations. From a large number of candidates, this technique is able to identify known core fragments responsible for primary recognition by the FK506 binding protein (FKBP-12), along with a diverse repertoire of novel molecular cores. By contrast, absolute energetic criteria for selecting molecular anchors are found to be promiscuous. A relationship between a minimum frustration principle of binding energy landscapes and receptor-specific molecular anchors in their role as "recognition nuclei" is established, thereby unraveling a mechanism of lead discovery and providing a practical route to receptor-biased computational combinatorial chemistry.

Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97.

The use of permeabilized cell models to study nuclear protein import has led to the identification of cytosolic components of the import machinery, including the NLS receptor, p97, Ran/TC4, and nuclear transport factor 2 (NTF2). These proteins are required to reconstitute docking of transport ligand at the nuclear pore complex and subsequent translocation through the nuclear pore. However, a detailed molecular understanding of how these factors mediate protein import is lacking. Here we describe the results of solution and solid phase binding assays, which demonstrate that the small GTPase Ran/TC4 interacts directly with the cytosolic transport factors p97 and NTF2. By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively. These data together with previous studies lead us to suggest that the interaction of the GTP-bound form of Ran/TC4 with p97 is linked to an early step in the nuclear protein import pathway and that the association of the GDP-bound form of Ran/TC4 with NTF2 helps define vectorial transport.

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.

Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.

Novel unconventional binding site in the variable region of immunoglobulins.

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

rSec6 and rSec8, mammalian homologs of yeast proteins essential for secretion.

Many of the molecules necessary for neurotransmission are homologous to proteins involved in the Golgi-to-plasma membrane stage of the yeast secretory pathway. Of 15 genes known to be essential for the later stages of vesicle trafficking in yeast, 7 have no identified mammalian homologs. These include the yeast SEC6, SEC8, and SEC15 genes, whose products are constituents of a 19.5S particle that interacts with the GTP-binding protein Sec4p. Here we report the sequences of rSec6 and rSec8, rat homologs of Sec6p and Sec8p. The rSec6 cDNA is predicted to encode an 87-kDa protein with 22% amino acid identity to Sec6p, and the rSec8 cDNA is predicted to encode a 110-kDa protein which is 20% identical to Sec8p. Northern blot analysis indicates that rSec6 and rSec8 are expressed in similar tissues. Immunodetection reveals that rSec8 is part of a soluble 17S particle in brain. COS cell cotransfection studies demonstrate that rSec8 colocalizes with the GTP-binding protein Rab3a and syntaxin 1a, two proteins involved in synaptic vesicle docking and fusion at the presynaptic terminal. These data suggest that rSec8 is a component of a high molecular weight complex which may participate in the regulation of vesicle docking and fusion in brain.

An Arabidopsis syntaxin homologue isolated by functional complementation of a yeast pep12 mutant.

The syntaxin family of integral membrane proteins are thought to function as receptors for transport vesicles, with different isoforms of this family localized to various membranes throughout the cell. The yeast Pep12 protein is a syntaxin homologue which may function in the trafficking of vesicles from the trans-Golgi network to the vacuole. We have isolated an Arabidopsis thaliana cDNA by functional complementation of a yeast pep12 mutant. The Arabidopsis cDNA (aPEP12) potentially encodes a 31-kDa protein which is homologous to yeast Pep12 and to other members of the syntaxin family, indicating that this protein may function in the docking or fusion of transport vesicles with the vacuolar membrane in plant cells. Northern blot analysis indicates that the mRNA is expressed in all tissues examined, although at a very low level in leaves. The mRNA is found in all cell types in roots and leaves, as shown by in situ hybridization experiments. The existence of plant homologues of proteins of the syntaxin family indicates that the basic vesicle docking and fusion machinery may be conserved in plants as it is in yeast and mammals.

The kinetics of quantal transmitter release from retinal amacrine cells.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.

Yeast synaptobrevin homologs are modified posttranslationally by the addition of palmitate.

Yeast possess two homologs of the synaptobrevin family of vesicle-associated membrane proteins that function in membrane recognition and vesicle fusion. Yeast proteins Snc1 and Snc2 localize to secretory vesicles and are required for constitutive exocytosis. They also form a physical complex with a plasma membrane protein, Sec9, which is necessary for vesicle docking and fusion to occur in vivo. Formation of this molecular complex, as a prerequisite for vesicle fusion, appears to have been conserved evolutionarily. Here we demonstrate that Snc proteins undergo a single posttranslational modification with the addition of a palmitate moiety to Cys-95 in Snc1. Modification of Cys-95 (which is located proximal to the transmembrane domain) is rapid, occurs in the endoplasmic reticulum, and is long-lasting. Mutation of Cys-95 to Ser-95 blocks palmitoylation and appears to affect Snc protein stability. This provides evidence that synaptobrevin-like proteins are modified posttranslationally, and we predict that fatty acylation may be common to those found in higher eukaryotes.

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.

Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine

Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (IACh) in a dose-dependent manner (IC50 close to 70 μM), but unlike lidocaine, DEA did not affect IACh desensitization. IACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced IACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and interphases, likely because of its larger size. Together, these results indicate that DEA mimics some, but not all, inhibitory actions of lidocaine on nAChRs and that even this small polar molecule acts by different mechanisms on this receptor. The presented results contribute to a better understanding of the structural determinants of nAChR modulation.

Cerebellar language and cognitive functions in childhood: A comparative review of the clinical research

Background: Recent research addressing evidence from functional neuroimaging studies, neurophysiological research, and new advances in neuropsychology together with traditional cerebellar lesion studies have recently implicated the cerebellum in adult language and cognitive functions. However, more limited information is currently available in describing the functional connectivity present in the paediatric population. Aims: It is the purpose of this paper to review recent clinical research pertaining to paediatric populations, outlining the impact of site of lesion and specific associated clinical changes in children with cerebellar disturbances. Main contribution: The specific contribution of the right cerebellar hemisphere to language function is identified to also exist in the paediatric population, highlighting the existence of functional connections between this region of the brain and left frontal cortical areas early in development. Conclusions: Implications for future research in paediatric populations are extensive, as a greater awareness and an understanding of the recently acknowledged involvement of the cerebellum in cognition and nonmotor linguistic function is anticipated to also add new dimension and direction to the analysis of childhood language outcomes associated with the cerebellum.