scholarly journals Allosteric stabilization of calcium and lipid binding engages three synaptotagmins in fast exocytosis

2021 ◽  
Author(s):  
Janus R. L. Kobbersmed ◽  
Manon M.M. Berns ◽  
Susanne Ditlevsen ◽  
Jakob B Sorensen ◽  
Alexander M Walter
Keyword(s):  
Full Range ◽  
Lipid Binding ◽  
Synaptic Function ◽  
Dominant Negative ◽  
Triggering Mechanism ◽  
Crucial Feature ◽  
Copy Numbers ◽  
Biochemical Measurements ◽  
Kinetics Of ◽  
Allosteric Properties

The release of neurotransmitters from presynaptic terminals is a strongly Ca2+-dependent process controlled by synaptotagmins, especially by their C2B domains. Biochemical measurements have reported Ca2+ affinities of synaptotagmin too low to account for synaptic function. However, binding of the C2B domain to the membrane phospholipid PI(4,5)P2 increases the Ca2+ affinity and vice versa, indicating a positive allosteric stabilization of simultaneous binding. Here, we construct a mathematical model of the release-triggering mechanism of synaptotagmin based on measured Ca2+/PI(4,5)P2 affinities and reported protein copy numbers. The model reproduced the kinetics of synaptic transmission observed at the calyx of Held over the full range of Ca2+ stimuli, with each C2B domain crosslinking Ca2+ and PI(4,5)P2 lowering the energy barrier for fusion by 4.85 kBT. The allosteric stabilization of simultaneous Ca2+ and PI(4,5)P2 binding was crucial to form multiple crosslinks which enabled fast fusion rates. Only three crosslinking C2B domains were needed to reproduce physiological responses, but high copy numbers per vesicle sped up the collision-limited formation of crosslinks. In silico evaluation of theoretical mutants revealed that affection of the allosteric properties might be a determinant of the severity of synaptotagmin mutations and may underlie dominant-negative, disease-causing effects. We conclude that allostericity is a crucial feature of synaptotagmin action.

scholarly journals A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

2007 ◽  
Vol 176 (3) ◽  
pp. 263-268 ◽  
Author(s):  
Adam C. Smith ◽  
Won Do Heo ◽  
Virginie Braun ◽  
Xiuju Jiang ◽  
Chloe Macrae ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Dominant Negative ◽  
Rab Gtpases ◽  
Wild Type ◽  
Intracellular Growth ◽  
Non Invasive ◽  
Serovar Typhimurium ◽  
Guanosine Triphosphatase ◽  
Kinetics Of

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

scholarly journals The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function

2015 ◽  
Vol 26 (18) ◽  
pp. 3275-3288 ◽  
Author(s):  
Kate Koles ◽  
Emily M. Messelaar ◽  
Zachary Feiger ◽  
Crystal J. Yu ◽  
C. Andrew Frank ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Postsynaptic Membrane ◽  
Lipid Binding ◽  
Synaptic Function ◽  
Muscle Membrane ◽  
Membrane Remodeling ◽  
Cellular Functions ◽  
Synaptic Homeostasis ◽  
Larval Neuromuscular Junction ◽  
Lipid Binding Proteins

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. Here, we show that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. Our results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR and uncover new roles for an EHD protein at synapses.

scholarly journals The ubiquitin E3 ligase POSH regulates calcium homeostasis through spatial control of Herp

2007 ◽  
Vol 177 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Shmuel Tuvia ◽  
Daniel Taglicht ◽  
Omri Erez ◽  
Iris Alroy ◽  
Iris Alchanati ◽  
...  
Keyword(s):  
Calcium Homeostasis ◽  
Critical Role ◽  
Dominant Negative ◽  
Spatial Control ◽  
Ubiquitin E3 Ligase ◽  
Lysine Residues ◽  
Domain Protein ◽  
Golgi Network ◽  
Kinetics Of

The ubiquitin (Ub) domain protein Herp plays a crucial role in the maintenance of calcium homeostasis during endoplasmic reticulum (ER) stress. We now show that Herp is a substrate as well as an activator of the E3 Ub ligase POSH. Herp-mediated POSH activation requires the Ubl domain and exclusively promotes lysine-63–linked polyubiquitination. Confocal microscopy demonstrates that Herp resides mostly in the trans-Golgi network, but, shortly after calcium perturbation by thapsigargin (Tpg), it appears mainly in the ER. Substitution of all lysine residues within the Ubl domain abolishes lysine-63–linked polyubiquitination of Herp in vitro and calcium-induced Herp relocalization that is also abrogated by the overexpression of a dominant-negative POSHV14A. A correlation exists between the kinetics of Tpg-induced Herp relocalization and POSH-dependent polyubiquitination. Finally, the overexpression of POSH attenuates, whereas the inhibition of POSH by the expression of POSHV14A or by RNA interference enhances Tpg-induced calcium burst. Altogether, these results establish a critical role for POSH-mediated ubiquitination in the maintenance of calcium homeostasis through the spatial control of Herp.

scholarly journals The Role of the Double Layer for the Pseudocapacitance of the Hydrogen Adsorption on Platinum

2022 ◽  
Author(s):  
Maximilian Schalenbach ◽  
Y. Emre Durmus ◽  
Hermann Tempel ◽  
Hans Kungl ◽  
Rüdiger-A. Eichel
Keyword(s):  
Double Layer ◽  
Hydrogen Adsorption ◽  
Full Range ◽  
Surface States ◽  
Alternative Interpretation ◽  
Kinetics Of Adsorption ◽  
Adsorption Processes ◽  
Kinetics Of ◽  
Cyclic Voltammetry Data

Abstract Pseudocapacitances such as the hydrogen adsorption on platinum (HAoPt) are associated with faradaic chemical processes that appear as capacitive in their potentiodynamic response, which was reported to result from the kinetics of adsorption processes. This study discusses an alternative interpretation of the partly capacitive response of the HAoPt that is based on the proton transport of ad- or desorbed hydrogen in the double layer. Potentiodynamic perturbations of equilibrated surface states of the HAoPt lead to typical double layer responses with the characteristic resistive-capacitive relaxations that overshadow the fast adsorption kinetics. A potential-dependent double layer representation by a dynamic transmission line model incorporates the HAoPt in terms of capacitive contributions and can computationally reconstruct the charge exchanged in full range cyclic voltammetry data. The coupling of charge transfer with double layer dynamics displays a novel physicochemical theory to explain the phenomenon of pseudocapacitance and the mechanisms in thereon based supercapacitors.

scholarly journals Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function

2016 ◽  
Vol 23 (11) ◽  
pp. 965-973 ◽  
Author(s):  
Baptiste Fischer ◽  
Kevin Lüthy ◽  
Jone Paesmans ◽  
Charlotte De Koninck ◽  
Ine Maes ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Lipid Binding ◽  
Synaptic Function

scholarly journals Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

Biosensors ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 145
Author(s):  
Antonio Guerrieri ◽  
Rosanna Ciriello ◽  
Giuliana Bianco ◽  
Francesca De Gennaro ◽  
Silvio Frascaro
Keyword(s):  
Trichoderma Viride ◽  
Kinetic Studies ◽  
Pt Electrode ◽  
Allosteric Enzyme ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Influence ◽  
Ph Dependent ◽  
Kinetics Of ◽  
Allosteric Properties

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

Type 1 von Willebrand disease mutation Cys1149Arg causes intracellular retention and degradation of heterodimers: a possible general mechanism for dominant mutations of oligomeric proteins

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 2973-2979 ◽  
Author(s):  
Imre Bodó ◽  
Akira Katsumi ◽  
Elodee A. Tuley ◽  
Jeroen C. J. Eikenboom ◽  
Zhengyu Dong ◽  
...  
Keyword(s):  
Full Range ◽  
Disease Type ◽  
Von Willebrand Disease ◽  
Dominant Negative ◽  
General Mechanism ◽  
Oligomeric Proteins ◽  
Dominant Phenotype ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Some families affected by von Willebrand disease type 1 show high penetrance with exceptionally low von Willebrand factor (VWF) levels. Previously, a mutation associated with this dominant phenotype, Cys1149Arg, was found to decrease the secretion of coexpressed normal VWF, and the mutation was proposed to cause intracellular retention of pro-VWF heterodimers. To demonstrate heterodimer formation, a model was developed in which subunits could be distinguished immunologically and by size. Recombinant VWF lacking domain A1 (dA1), A3 (dA3), or both (dA13) was secreted efficiently as a full range of multimers. Cotransfection of Cys1149Arg and dA13 resulted in the secretion of multimeric VWF containing about 250 kd (Cys1149Arg) and about 210 kd (dA13). Cell lysates contained pro-VWF forms of Cys1149Arg and dA13. Immunoprecipitation with an antidomain A1 antibody recovered both subunits in heterodimers, and subunit ratios were consistent with random dimerization. Similar results were obtained for cotransfection of Cys1149Arg and dA1. Normal VWF has a Cys1149-Cys1169 intrachain bond. When cotransfected with normal VWF, Cys1149Arg or the double mutant Cys1149Arg+Cys1169Ser caused a similar decrease in VWF secretion, suggesting that an unpaired Cys1169 does not explain the intracellular retention of Cys1149Arg. VWF Cys1149Arg was not secreted from BHK cells but was degraded intracellularly within about 4 hours, and the proteasome inhibitor lactacystin delayed its clearance more than 16 hours. Thus, dominant von Willebrand disease type 1 may be caused by heterodimerization of mutant and normal subunits in the endoplasmic reticulum followed by proteasomal degradation in the cytoplasm. A similar dominant negative mechanism could cause quantitative deficiencies of other multisubunit proteins.

scholarly journals Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton

2002 ◽  
Vol 157 (3) ◽  
pp. 469-479 ◽  
Author(s):  
Mireille Lambert ◽  
Daniel Choquet ◽  
René-Marc Mège
Keyword(s):  
Actin Cytoskeleton ◽  
Optical Tweezers ◽  
Cell Sorting ◽  
Single Particle Tracking ◽  
Dominant Negative ◽  
Cell Contact ◽  
Ligand Density ◽  
Dominant Negative Form ◽  
Kinetics Of ◽  
Negative Form

Cadherin receptors are key morphoregulatory molecules during development. To dissect their mode of action, we developed an approach based on the use of myogenic C2 cells and beads coated with an Ncad-Fc ligand, allowing us to mimic cadherin-mediated adhesion. We used optical tweezers and video microscopy to investigate the dynamics of N-cadherin anchoring within the very first seconds of bead–cell contact. The analysis of the bead movement by single-particle tracking indicated that N-cadherin molecules were freely diffusive in the first few seconds after bead binding. The beads rapidly became diffusion-restricted and underwent an oriented rearward movement as a result of N-cadherin anchoring to the actin cytoskeleton. The kinetics of anchoring were dependent on ligand density, suggesting that it was an inducible process triggered by active cadherin recruitment. This anchoring was inhibited by the dominant negative form of Rac1, but not that of Cdc42. The Rac1 mutant had no effect on cell contact formation or cadherin–catenin complex recruitment, but did inhibit actin recruitment. Our results suggest that cadherin anchoring to the actin cytoskeleton is an adhesion-triggered, Rac1-regulated process enabling the transduction of mechanical forces across the cell membrane; they uncover novel aspects of the action of cadherins in cell sorting, cell migration, and growth cone navigation.

scholarly journals Human Papillomavirus E6 Regulates the Cytoskeleton Dynamics of Keratinocytes through Targeted Degradation of p53

2007 ◽  
Vol 81 (22) ◽  
pp. 12675-12679 ◽  
Author(s):  
Brooke Cooper ◽  
Nicole Brimer ◽  
Scott B. Vande Pol
Keyword(s):  
Human Papillomavirus ◽  
Cell Spreading ◽  
Dominant Negative ◽  
Bovine Papillomavirus ◽  
Human Papillomavirus Type 16 ◽  
Extracellular Matrix Molecules ◽  
Cervical Cells ◽  
High Risk Hpv ◽  
Cytoskeleton Dynamics ◽  
Kinetics Of

ABSTRACT The attachment and spreading of keratinocyte cells result from interactions between integrins and immobilized extracellular matrix molecules. Human papillomavirus type 16 (HPV-16) E6 augmented the kinetics of cell spreading, while E6 genes from HPV-11 or bovine papillomavirus type 1 did not. The ability of E6 to interact with the E6AP ubiquitin ligase and target p53 degradation was required to augment cell-spreading kinetics; dominant negative p53 alleles also enhanced the kinetics of cell spreading and the level of attachment of cells to hydrophobic surfaces. The targeted degradation of p53 by E6 may contribute to the invasive phenotype exhibited by cervical cells that contain high-risk HPV types.

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