An ultra-stable cytoplasmic antibody engineered for in vivo applications.

Targeting cytoplasmic protein-protein interactions with antibodies remains technically challenging, since antibodies expressed in the cytosol frequently form insoluble aggregates. Existing engineering methods are based on the notion that the estimated net charge at pH 7.4 affects stability; as such, they are unable to overcome this problem. Herein, we report a versatile method for engineering an ultra-stable cytoplasmic antibody (STAND), with a strong estimated net negative charge at pH 6.6, by fusing peptide tags with a highly negative charge and a low isoelectric point. Without the need for complicated amino acid substitutions, we convert aggregation-prone antibodies to STANDs that are useful for inhibiting in vivo transmitter release, modulating animal behaviour, and inhibiting in vivo cancer proliferation driven by mutated Kras-long recognised as an "undruggable" oncogenic protein. The STAND method shows promise for targeting endogenous cytoplasmic proteins in basic biology and for developing future disease treatments.

Parkin promotes proteasomal degradation of synaptotagmin IV by accelerating polyubiquitination.

Parkin is an E3 ubiquitin ligase whose mutations cause autosomal recessive juvenile Parkinson's disease (PD). Unlike the human phenotype, parkin knockout (KO) mice show no apparent dopamine neuron degeneration, although they demonstrate reduced expression and activity of striatal mitochondrial proteins believed to be necessary for neuronal survival. Instead, parkin-KO mice show reduced striatal evoked dopamine release, abnormal synaptic plasticity, and non-motor symptoms, all of which appear to mimic the preclinical features of Parkinson's disease. Extensive studies have screened candidate synaptic proteins responsible for reduced evoked dopamine release, and synaptotagmin XI (Syt XI), an isoform of Syt family regulating membrane trafficking, has been identified as a substrate of parkin in humans. However, its expression level is unaltered in the striatum of parkin-KO mice. Thus, the target(s) of parkin and the molecular mechanisms underlying the impaired dopamine release in parkin-KO mice remain unknown. In this study, we focused on Syt IV because of its highly homology to Syt XI, and because they share an evolutionarily conserved lack of Ca2+-binding capacity; thus, Syt IV plays an inhibitory role in Ca2+-dependent neurotransmitter release in PC12 cells and neurons in various brain regions. We found that a proteasome inhibitor increased Syt IV protein, but not Syt XI protein, in neuron-like, differentiated PC12 cells, and that parkin interacted with and polyubiquitinated Syt IV, thereby accelerating its protein turnover. Parkin overexpression selectively degraded Syt IV protein, but not Syt I protein (indispensable for Ca2+-dependent exocytosis), thus enhancing depolarization-dependent exocytosis. Furthermore, in parkin-KO mice, the level of striatal Syt IV protein was increased. Our data indicate a crucial role for parkin in the proteasomal degradation of Syt IV, and provide a potential mechanism of parkin-regulated, evoked neurotransmitter release.

Syntaxin 1B suppresses macropinocytosis and semaphorin 3A-induced growth cone collapse.

Growth cone collapse is a crucial process for repulsive axon guidance and is accompanied by a reduction in growth cone surface area. This process of reduction may be regulated by endocytosis; however, its molecular mechanism is unclear. Macropinocytosis is a clathrin-independent form of endocytosis in which large areas of plasma membrane can be engulfed. We have reported previously that macropinocytosis is induced in growth cones of chick dorsal root ganglion neurons by semaphorin 3A (Sema3A), a repulsive axon guidance cue, and that Sema3A-induced reduction in growth cone surface area and macropinocytic vacuole area were correlated, suggesting a positive role for macropinocytosis in Sema3A-induced growth cone collapse. In the present study, we found that syntaxin 1B (Syx1B), a membrane trafficking protein, is a negative regulator of macropinocytosis, and its expression is downregulated by Sema3A signaling. Macropinocytosis inhibitor ethylisopropylamiloride or Syx1B overexpression suppressed Sema3A-induced macropinocytosis and growth cone collapse. These results indicate that Syx1B couples macropinocytosis-mediated massive internalization of the plasma membrane to Sema3A-induced growth cone collapse.

Dissociation of inositol polyphosphates from the C2B domain of synaptotagmin facilitates spontaneous release of catecholamines in adrenal chromaffin cells. A suggestive evidence of a fusion clamp by synaptotagmin.

Synaptotagmins (Syts) serve as a Ca²+ sensor in the release of neurotransmitters and hormones. Inositol polyphosphates (InsPPs) such as Inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6) bind to Ca²+-binding C2B domain of Syt I and II, and inhibit transmitter release. We have shown that the inhibition by InsPPs is reversed by Ca²+ in adrenal chromaffin cells, while a rapid accumulation of endogenous InsP5 and InsP6 upon depolarizing stimuli have been reported in these and some other cells. Such a rapid accumulation of InsPPs, if not all, might reflect their dissociation from C2B domain of Syt. To elucidate the functional relevance, we studied the effects of antibodies against C2A and C2B domains (anti-C2A Ab, anti-C2B Ab) on the accumulation of InsPPs induced by Ca²+ in digitonin-permeabilized adrenal chromaffin cells. Anti-C2B Ab by itself caused an accumulation of InsPPs in the permeabilizing medium, and increased spontaneous release of catecholamines (CA). Anti-C2A Ab abolished Ca²+-induced increase of InsPPs in cytosolic component, and inhibited Ca²+-evoked release of CA with little effect on the spontaneous release. Microinjection of InsP6 but not inositol hexakissulfate into intact chromaffin cells inhibited both spontaneous and nicotine-evoked exocytotic events. These results suggest that endogenous InsPPs bound to the C2B domain clamp spontaneous fusion of the docked or primed vesicles at resting level of intracellular Ca²+, and binding of Ca²+ to the C2A or/and C2B domain facilitate fusion dissociating InsPPs from Syt in adrenal chromaffin cells. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Inositol 1, 4, 5-trisphosphate receptor interacts with the SNARE domain of syntaxin 1B.

Inositol 1, 4, 5-trisphosphate receptors (IP(3)Rs) are intracellular ligand-gated Ca(2+) channels that mediate Ca(2+) release from the endoplasmic reticulum (ER) into the cytosol and function in diverse cellular processes including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. The Ca(2+) release activity of IP(3)Rs is tightly regulated by many factors including IP(3)R-binding proteins. We show that IP(3)Rs interact with syntaxin 1 (Syx1), a membrane trafficking protein that regulates various plasma-membrane ion channels including N-, P/Q, and L-type voltage-gated Ca(2+) channels, voltage-gated potassium channels, and an epithelial sodium channel. We found that a SNARE-domain of Syx1B, one of the two Syx1 isoforms, directly interacted with the type1 IP(3)R (IP(3)R1) internal coupling domain, a known modulator for channel opening. These results indicate that Syx1B is an IP(3)R-interacting protein and that its interaction may play a crucial role in regulating the channel activity of IP(3)Rs in Syx1B-expressing cells.

Ca2+ induces macropinocytosis via F-actin depolymerization during growth cone collapse.

Growth cone collapse occurs in repulsive axon guidance and is accompanied by a reduction in the surface area of the plasma membrane of growth cones. However, the mechanism of this reduction is unclear. Here, we show that during growth cone collapse, caffeine-induced Ca(2+) release from ryanodine-sensitive Ca(2+) stores triggers the formation of large vacuoles in growth cones by macropinocytosis, a clathrin-independent endocytosis for the massive retrieval of the cellular plasma membrane, and subsequent retrograde membrane transport. We observed a significant correlation of the area of caffeine-induced macropinosomes with growth cone collapse. We also detected macropinocytosis induced by semaphorin 3A, a typical repulsive cue, and correlation between the area of semaphorin 3A-induced macropinocytic vacuoles and growth cone collapse. Moreover, jasplakinolide, an inhibitor of F-actin depolymerization, blocked caffeine-induced macropinocytosis. We propose that the coordinated regulation of actin cytoskeletal reorganization and macropinocytosis-mediated retrograde membrane trafficking may contribute to Ca(2+)-induced axon growth inhibition.

Syntaxin 6 regulates nerve growth factor-dependent neurite outgrowth.

Neurite outgrowth is crucial for neural circuit formation. Intracellular membrane trafficking is involved in the cell surface expansion that is necessary for neurite outgrowth. It is known that syntaxin 6 is predominantly located in the Golgi region in undifferentiated PC12 cells and that it regulates trans-Golgi network trafficking and the secretory pathway via its coiled-coil domains. However, whether it also regulates neurite outgrowth remains unknown. In this paper, we found that syntaxin 6 was located both in the Golgi apparatus and the distal tips of the neurites of nerve growth factor (NGF)-treated PC12 cells. We also showed that the overexpression of the first coiled-coil domain of syntaxin 6 inhibited NGF-dependent neurite outgrowth. However, the coiled-coil domain-disrupting mutant had little effect on neurite outgrowth. These results suggest that the first coiled-coil domain of syntaxin 6 plays a crucial role in NGF-dependent neurite outgrowth.