SUMO: In Silico Sequence Assessment Using Multiple Optimization Parameters.

To select the most promising screening hits from antibody and VHH display campaigns for subsequent in-depth profiling and optimization, it is highly desirable to assess and select sequences on properties beyond only their binding signals from the sorting process. In addition, developability risk criteria, sequence diversity, and the anticipated complexity for sequence optimization are relevant attributes for hit selection and optimization. Here, we describe an approach for the in silico developability assessment of antibody and VHH sequences. This method not only allows for ranking and filtering multiple sequences with regard to their predicted developability properties and diversity, but also visualizes relevant sequence and structural features of potentially problematic regions and thereby provides rationales and starting points for multi-parameter sequence optimization.

Allosteric modulation of a human odorant receptor.

Odor perception is first determined by how the myriad of environmental volatiles are detected at the periphery of the olfactory system. The combinatorial activation of dedicated odorant receptors generates enough encoding power for the discrimination of tens of thousands of odorants. Recent studies have revealed that odorant receptors undergo widespread inhibitory modulation of their activity when presented with mixtures of odorants, a property likely required to maintain discrimination and ensure sparsity of the code for complex mixtures. Here, we establish the role of human OR5AN1 in the detection of musks and identify distinct odorants capable of enhancing its activity in binary mixtures. Chemical and pharmacological characterization indicate that specific α-ß unsaturated aliphatic aldehydes act as positive allosteric modulators. Sensory experiments show decreased odor detection threshold in humans, suggesting that allosteric modulation of odorant receptors is perceptually relevant and likely adds another layer of complexity to how odors are encoded in the peripheral olfactory system.

Syntaxins on granules promote docking of granules via interactions with munc18.

SNAREs and SNARE-binding accessory proteins are believed to be central molecular components of neurotransmitter release, although the precise sequence of molecular events corresponding to distinct physiological states is unclear. The mechanism of docking of vesicles to the plasma membrane remains elusive, as the anchoring protein residing on vesicles is unknown. Here I show that targeting small amounts of syntaxin to granules by transmembrane domain alteration leads to a substantial enhancement of syntaxin clustering beneath granules, as well as of morphological granule docking. The effect was abolished without munc18 and strongly reduced by removal of the N-terminal peptide in the syntaxin mutant. Thus, in contrast to the current paradigm, I demonstrate that syntaxin acts from the vesicular membrane, strongly facilitating docking of vesicles, likely via interaction of its N-peptide with munc18. Docking was assayed by quantifying the syntaxin clusters beneath granules, using two-color Total Internal Reflectance Fluorescence microscopy in live PC-12 cells and confirmed by electron microscopy. Hereby, I propose a new model of vesicle docking, wherein munc18 bridges the few syntaxin molecules residing on granules to the syntaxin cluster on the plasma membrane, suggesting that the number of syntaxins on vesicles determines docking and conceivably fusion probability.

Dual-transmitter neurons: functional implications of co-release and co-transmission.

Co-transmission, the ability of a neuron to release multiple transmitters, has long been recognized in selected circuits. However, the release of multiple primary neurotransmitters from a single neuron is only beginning to be appreciated. Here we consider recent examples of co-transmission as well as co-release-the packaging of multiple neurotransmitters into a single vesicle. The properties associated with each mode of release greatly enhance the possible action of such neurons within circuits. The functional importance of dual- (or multi-) transmitter neurons extends beyond actions on postsynaptic receptors, due in part to differential spatial and temporal profiles of each neurotransmitter. Recent evidence also suggests that the dual-transmitter phenotype can be dynamically regulated during development and following injury or disease.

Distinct modes of dopamine and GABA release in a dual transmitter neuron.

We now know of a surprising number of cases where single neurons contain multiple neurotransmitters. Neurons that contain a fast-acting neurotransmitter, such as glutamate or GABA, and a modulatory transmitter, such as dopamine, are a particularly interesting case because they presumably serve dual signaling functions. The olfactory bulb contains a large population of GABA- and dopamine-containing neurons that have been implicated in normal olfaction as well as in Parkinson's disease. Yet, they have been classified as nonexocytotic catecholamine neurons because of the apparent lack of vesicular monoamine transporters. Thus, we examined how dopamine is stored and released from tyrosine hydroxylase-positive GFP (TH(+)-GFP) mouse periglomerular neurons in vitro. TH(+) cells expressed both VMAT2 (vesicular monoamine transporter 2) and VGAT (vesicular GABA transporter), consistent with vesicular storage of both dopamine and GABA. Carbon fiber amperometry revealed that release of dopamine was quantal and calcium-dependent, but quantal size was much less than expected for large dense core vesicles, suggesting that release originated from small clear vesicles identified by electron microscopy. A single action potential in a TH(+) neuron evoked a brief GABA-mediated synaptic current, whereas evoked dopamine release was asynchronous, lasting for tens of seconds. Our data suggest that dopamine and GABA serve temporally distinct roles in these dual transmitter neurons.

Membrane-proximal tryptophans of synaptobrevin II stabilize priming of secretory vesicles.

Trans-soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complexes formed between the SNARE motifs of synaptobrevin II, SNAP-25, and syntaxin play an essential role in Ca(2+)-regulated exocytosis. Apart from the well studied interactions of the SNARE domains, little is known about the functional relevance of other evolutionarily conserved structures in the SNARE proteins. Here, we show that substitution of two highly conserved tryptophan residues within the juxtamembrane domain (JMD) of the vesicular SNARE Synaptobrevin II (SybII) profoundly impairs priming of granules in mouse chromaffin cells without altering catecholamine release from single vesicles. Using molecular dynamic simulations of membrane-embedded SybII, we show that Trp residues of the JMD influence the electrostatic surface potential by controlling the position of neighboring lysine and arginine residues at the membrane-water interface. Our observations indicate a decisive role of the tryptophan moiety of SybII in keeping the vesicles in the release-ready state and support a model wherein tryptophan-mediated protein-lipid interactions assist in bridging the apposing membranes before fusion.

Loss of olfactory cell adhesion molecule reduces the synchrony of mitral cell activity in olfactory glomeruli.

Odours generate activity in olfactory receptor neurons, whose axons contact the dendritic tufts of mitral cells within olfactory bulb glomeruli. These axodendritic synapses are anatomically separated from dendrodendritic synapses within each glomerulus. Mitral cells within a glomerulus show highly synchronized activity as assessed with whole-cell recording from pairs of mitral cells. We examined glomerular activity in mice lacking the olfactory cell adhesion molecule (OCAM). Glomeruli in mice lacking OCAM show a redistribution of synaptic subcompartments, but the total area occupied by axonal inputs was similar to wild-type mice. Stimulation of olfactory nerve bundles showed that excitatory synaptic input to mitral cells as well as dendrodendritic inhibition was unaffected in the knockout. However, correlated spiking in mitral cells was significantly reduced, as was electrical coupling between apical dendrites. To analyse slow network dynamics we induced slow oscillations with a glutamate uptake blocker. Evoked and spontaneous slow oscillations in mitral cells and external tufted cells were broader and had multiple peaks in OCAM knockout mice, indicating that synchrony of slow glomerular activity was also reduced. To assess the degree of shared activity between mitral cells under physiological conditions, we analysed spontaneous sub-threshold voltage oscillations using coherence analysis. Coherent activity was markedly reduced in cells from OCAM knockout mice across a broad range of frequencies consistent with a decrease in tightly time-locked activity. We suggest that synchronous activity within each glomerulus is dependent on segregation of synaptic subcompartments.

v-SNARE actions during Ca(2+)-triggered exocytosis.

Assembly of SNARE proteins between opposing membranes mediates fusion of synthetic liposomes, but it is unknown whether SNAREs act during exocytosis at the moment of Ca(2+) increase, providing the molecular force for fusion of secretory vesicles. Here, we show that execution of pre- and postfusional steps during chromaffin granule exocytosis depends crucially on a short molecular distance between the complex-forming SNARE motif and the transmembrane anchor of the vesicular SNARE protein synaptobrevin II. Extending the juxtamembrane region of synaptobrevin by insertion of flexible "linkers" reduces priming of granules, delays initiation of exocytosis upon stepwise elevation of intracellular calcium, attenuates fluctuations of early fusion pores, and slows rapid expansion of the pore in a linker-length dependent fashion. These observations provide evidence that v-SNARE proteins drive Ca(2+)-triggered membrane fusion at millisecond time scale and support a model wherein continuous molecular pulling by SNAREs guides the vesicle throughout the consecutive stages of exocytosis.

v-SNAREs control exocytosis of vesicles from priming to fusion.

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.