scholarly journals Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion

2016 ◽  
Vol 113 (38) ◽  
pp. 10536-10541 ◽  
Author(s):  
Feng Li ◽  
Neeraj Tiwari ◽  
James E. Rothman ◽  
Frederic Pincet
Keyword(s):  
Activation Energy ◽  
Energy Barrier ◽  
Vesicle Docking ◽  
Readily Releasable Pool ◽  
Protein Receptor ◽  
Regulatory Machinery ◽  
Presynaptic Plasma Membrane ◽  
Kinetic Barriers

Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.

scholarly journals Mechanism of CK2.3, a Novel Mimetic Peptide of Bone Morphogenetic Protein Receptor Type IA, Mediated Osteogenesis

2019 ◽  
Vol 20 (10) ◽  
pp. 2500 ◽  
Author(s):  
Vrathasha Vrathasha ◽  
Hilary Weidner ◽  
Anja Nohe
Keyword(s):  
Signaling Pathways ◽  
Treatment Options ◽  
Time Frame ◽  
Bmp Signaling ◽  
C2c12 Cells ◽  
Molecular Sequence ◽  
Sequence Of Events ◽  
Protein Receptor

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle

2007 ◽  
Vol 292 (6) ◽  
pp. L1473-L1479 ◽  
Author(s):  
Moira Hagen ◽  
Karen Fagan ◽  
Wolfgang Steudel ◽  
Michelle Carr ◽  
Kirk Lane ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transgenic Mice ◽  
Bmp Signaling ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Second Hit ◽  
Protein Receptor ◽  
Bmp Pathway

The majority of familial pulmonary arterial hypertension (PAH) cases are caused by mutations in the type 2 bone morphogenetic protein receptor (BMPR2). However, less than one-half of BMPR2 mutation carriers develop PAH, suggesting that the most important function of BMPR2 mutation is to cause susceptibility to a “second hit.” There is substantial evidence from the literature implicating dysregulated inflammation, in particular the cytokine IL-6, in the development of PAH. We thus hypothesized that the BMP pathway regulates IL-6 in pulmonary tissues and conversely that IL-6 regulates the BMP pathway. We tested this in vivo using transgenic mice expressing an inducible dominant negative BMPR2 in smooth muscle, using mice injected with an IL-6-expressing virus, and in vitro using small interfering RNA (siRNA) to BMPR2 in human pulmonary artery smooth muscle cells (PA SMC). Consistent with our hypothesis, we found upregulation of IL-6 in both the transgenic mice and in cultured PA SMC with siRNA to BMPR2; this could be abolished with p38MAPK inhibitors. We also found that IL-6 in vivo caused a twofold increase in expression of the BMP signaling target Id1 and caused increased BMP activity in a luciferase-reporter assay in PA SMC. Thus we have shown both in vitro and in vivo a complete negative feedback loop between IL-6 and BMP, suggesting that an important consequence of BMPR2 mutations may be poor regulation of cytokines and thus vulnerability to an inflammatory second hit.

scholarly journals Positive and Negative Regulation of a SNARE Protein by Control of Intracellular Localization

2004 ◽  
Vol 15 (4) ◽  
pp. 1802-1815 ◽  
Author(s):  
Hideki Nakanishi ◽  
Pablo de los Santos ◽  
Aaron M. Neiman
Keyword(s):  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Intracellular Localization ◽  
Negative Regulation ◽  
Snare Protein ◽  
Nuclear Targeting ◽  
Protein Receptor ◽  
Prospore Membrane

In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.

scholarly journals Thermal inactivation and reactivation of an enzyme in vivo. Pantothenate hydrolase of Pseudomonas fluorescens

1972 ◽  
Vol 130 (1) ◽  
pp. 111-119 ◽  
Author(s):  
R. Kalervo Airas
Keyword(s):  
Activation Energy ◽  
Pseudomonas Fluorescens ◽  
Rapid Growth ◽  
Thermal Inactivation ◽  
Fast Rate ◽  
Carbon Sources ◽  
Enzyme Inactivation ◽  
Whole Cells

Thermal inactivation and reactivation of pantothenate hydrolase were studied in whole cells of Pseudomonas fluorescens. The enzyme is susceptible to thermal inactivation in whole cells at 37–40°C, and is reactivated when the temperature is lowered again. Chloramphenicol does not prevent reactivation. The activation energy of enzyme inactivation in vivo is about 540kJ/mol. This activation energy is 220kJ/mol in vitro, but it is increased to 550–630kJ/mol by several metabolites, such as succinate, glyoxylate and oxalate. Generally, good carbon sources, causing rapid growth, protect the enzyme from thermal inactivation in vivo, and enable reactivation to occur at a fast rate. The enzyme is also inactivated below 35°C, showing an activation energy of about 35kJ/mol. Good carbon sources prevent this inactivation as well, and cause slight reactivation. Glycine, although not utilized for growth, protects the enzyme well from this inactivation but not from inactivation at 37–40°C, and prevents reactivation totally. From the activation energies of inactivation and the effects of the various carbon sources, it appears possible that changes in the concentrations of intracellular metabolites may be responsible for the changes in inactivation and reactivation.

scholarly journals Mechanoresponsive Smad5 Enhances MiR-487a Processing to Promote Vascular Endothelial Proliferation in Response to Disturbed Flow

2021 ◽  
Vol 9 ◽  
Author(s):  
Wei-Li Wang ◽  
Li-Jing Chen ◽  
Shu-Yi Wei ◽  
Yu-Tsung Shih ◽  
Yi-Hsuan Huang ◽  
...  
Keyword(s):  
Shear Stress ◽  
Luciferase Reporter ◽  
Brdu Incorporation ◽  
Creb Binding Protein ◽  
Disturbed Flow ◽  
Argonaute 2 ◽  
Protein Receptor ◽  
Incorporation Assay

MicroRNAs (miRs) and bone morphogenetic protein receptor–specific Smads are mechano-responsive molecules that play vital roles in modulating endothelial cell (EC) functions in response to blood flow. However, the roles of interplay between these molecules in modulating EC functions under flows remain unclear. We elucidated the regulatory roles of the interplay between miR-487a and Smad5 in EC proliferation in response to different flow patterns. Microarray and quantitative RT-PCR showed that disturbed flow with low and oscillatory shear stress (OS, 0.5 ± 4 dynes/cm2) upregulates EC miR-487a in comparison to static controls and pulsatile shear stress (12 ± 4 dynes/cm2). MiR-487a expression was higher in ECs in the inner curvature (OS region) than the outer curvature of the rat aortic arch and thoracic aorta and also elevated in diseased human coronary arteries. MiR-487a expression was promoted by nuclear phospho-Smad5, which bound to primary-miR-487a to facilitate miR-487a processing. Algorithm prediction and luciferase reporter and argonaute 2-immunoprecipitation assays demonstrated that miR-487a binds to 3′UTR of CREB binding protein (CBP) and p53. Knockdown and overexpression of miR-487a decreased and increased, respectively, phospho-Rb and cyclin A expressions through CBP and p53. A BrdU incorporation assay showed that miR-487a enhanced EC proliferation under OS in vitro and in disturbed flow regions of experimentally stenosed rat abdominal aorta in vivo. These results demonstrate that disturbed flow with OS induces EC expression of miR-487a through its enhanced processing by activated-Smad5. MiR-487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR-487 may serve as an important molecular target for intervention against disturbed flow–associated vascular disorders resulting from atherosclerosis.

scholarly journals But Mouse, you are not alone: On some severe acute respiratory syndrome coronavirus 2 variants infecting mice

2021 ◽  
Author(s):  
Michael J Kuiper ◽  
Laurence Wilson ◽  
Shruthi Mangalaganesh ◽  
Daniel Reti ◽  
Seshadri S Vasan
Keyword(s):  
Adaptive Mutation ◽  
Aromatic Substitution ◽  
Angiotensin Converting Enzyme 2 ◽  
Protein Receptor ◽  
In Situ Observations ◽  
Animal Reservoirs ◽  
Do So

In silico predictions combined with in vitro, in vivo and in situ observations collectively suggest that mouse adaptation of the SARS-CoV-2 virus requires an aromatic substitution in position 501 or position 498 (but not both) of the spike protein receptor binding domain. This effect could be enhanced by mutations in positions 417, 484 and 493 (especially K417N, E484K, Q493K and Q493R), and to a lesser extent by mutations in positions 486 and 499 (such as F486L and P499T). Such enhancements due to more favourable binding interactions with residues on the complementary angiotensin-converting enzyme 2 (ACE2) interface, are however, unlikely to sustain mouse infectivity on their own based on theoretical and experimental evidence to date. Our current understanding thus points to the Alpha, Beta and Gamma variants of concern infecting mice, while Delta and Delta Plus lack a similar biomolecular basis to do so. This paper identifies a list of countries where local field surveillance of mice is encouraged because they may have come in contact with humans who had the virus with adaptive mutation(s). It also provides a systematic methodology to analyze the potential for other animal reservoirs and their likely locations.

scholarly journals Altered synaptic adaptation and gain in sensory circuits of the casein kinase 1 delta (CK1dT44A) mouse model of migraine

2019 ◽  
Author(s):  
Pratyush Suryavanshi ◽  
Punam Sawant Pokam ◽  
KC Brennan
Keyword(s):  
Steady State ◽  
Cortical Neurons ◽  
Casein Kinase ◽  
Local Network ◽  
Casein Kinase 1 ◽  
Thermal Pain ◽  
Readily Releasable Pool ◽  
Short Stimulus

AbstractMigraine is a very common and disabling neurological disorder that remains poorly understood at the cellular and circuit level. Transgenic mice harboring a mutation in casein kinase 1 delta (CK1dT44A) represent the first animal model of non-hemiplegic migraine. These mice have decreased sensory thresholds to mechanical and thermal pain after treatment with the migraine trigger nitroglycerin; and an increased susceptibility to cortical spreading depression (CSD), which models the migraine aura. In this study, we investigated cellular and synaptic mechanisms within sensory cortical circuits that might underlie the migraine relevant phenotypes of CK1dT44A mice, using in vitro and in vivo whole cell electrophysiology. Surprisingly we found that at resting state, CK1dT44A neurons exhibited hyperpolarized membrane potentials, due to increased tonic inhibition. Despite this reduction in baseline excitability, CK1dT44A neurons fired action potentials more frequently in response to current injection. And despite similar synaptic and dendritic characteristics to wild type neurons, excitatory but not inhibitory CK1dT44A synapses failed to adapt to high frequency short-stimulus trains, resulting in elevated steady state excitatory currents. The increased steady state currents were attributable to an increased replenishment rate of the readily releasable pool, providing a presynaptic mechanism for the CK1dT44A phenotype. Finally, during in vivo experiments, CK1dT44A animals showed increased duration and membrane potential variance at ‘cortical up states’, showing that the intrinsic and synaptic changes we observed have excitatory consequences at the local network level. In conclusion excitatory sensory cortical neurons and networks in CK1dT44A animals appear to exhibit decreased adaptation and increased gain that may inform the migraine phenotype.

scholarly journals SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion

1999 ◽  
Vol 339 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Carmen GONELLE-GISPERT ◽  
Philippe A. HALBAN ◽  
Heiner NIEMANN ◽  
Michael PALMER ◽  
Stefan CATSICAS ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
B Cells ◽  
Resistant Mutant ◽  
Secreting Cell ◽  
Protein Receptor ◽  
Target Membrane ◽  
Insulin Secreting Cells ◽  
Hit Cells

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R176QIDRIM182) was changed to P176QIKRIT182. This is the sequence of the equivalent region of human SNAP-23 (P187–T194), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E in vitro and in vivo and both mutant isoforms were able to reconstitute insulin secretion from toxin-treated HIT cells.

scholarly journals Reduced O-GlcNAcylation of SNAP-23 promotes cisplatin resistance by inducing exosome secretion in ovarian cancer

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Luomeng Qian ◽  
Xiaoshan Yang ◽  
Shaohui Li ◽  
Hang Zhao ◽  
Yunge Gao ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Protein Modification ◽  
Snare Complex ◽  
Ovarian Cancer Cells ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor

AbstractExosomes have been associated with chemoresistance in various cancers, but such a role in ovarian cancer is not yet clear. Here, using in vitro cell-based and in vivo mouse model experiments, we show that downregulation of O-GlcNAcylation, a key post-translational protein modification, promotes exosome secretion. This increases exosome-mediated efflux of cisplatin from cancer cells resulting in chemoresistance. Mechanistically, our data indicate that downregulation of O-GlcNAclation transferase (OGT) reduces O-GlcNAclation of SNAP-23. Notably, O-GlcNAcylation of SNAP-23 is vital for regulating exosome release in ovarian cancer cells. Reduced O-GlcNAclation of SNAP-23 subsequently promotes the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of SNAP-23, VAMP8, and Stx4 proteins. This enhances exosome release causing chemoresistance by increasing the efflux of intracellular cisplatin.

Chloride distribution and exchange in rat ventricle

1980 ◽  
Vol 238 (5) ◽  
pp. C169-C176 ◽  
Author(s):  
P. I. Polimeni ◽  
E. Page
Keyword(s):  
Activation Energy ◽  
Equilibrium Potential ◽  
Quasi Steady State ◽  
Cell Water ◽  
Analytical Error ◽  
Rat Ventricle ◽  
Calculated Equilibrium ◽  
Exchange Flux

The cellular Cl content and concentration ([Cl]cell) and the cellular uptake of 36Cl have been measured in the rat left ventricle in vivo. The in vitro efflux of 36Cl from perfused contracting ventricles preequilibrated with 36Cl in vivo was also determined at 22, 30, and 38 degrees C. [Cl]cell was 8.2 +/- 0.5 mmol/kg cell water, corresponding to a calculated equilibrium potential of Cl of -70 to -80 mV. This figure for [Cl]cell is significantly lower than previous estimates in the literature, which were subject to an analytical error leading to overestimation of muscle Cl content obtained coulometrically. At 38 degrees C, Cl exchange under quasi-steady-state conditions was 31.2 mumol . (g dry ventricle . min)-1 or 42.5 pmol . (cm2 plasma membrane . s).-1 Apparent activation energy of the flux was 10.4 kcal/mol. At 22 degrees C, no dependence of the exchange on contraction frequency was detectable over a range of 80-160 contractions/min. The Cl exchange flux is among the fastest, if not the fastest. known for myocardial ion transport.

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