Sec17 (α-SNAP) and an SM-tethering complex control the outcome of SNARE zippering in vitro and in vivo

Mapping Intimacies ◽

10.1101/123133 ◽

2017 ◽

Author(s):

Matthew L. Schwartz ◽

Daniel P. Nickerson ◽

Braden T. Lobingier ◽

Cortney G. Angers ◽

Michael Zick ◽

...

Keyword(s):

Early Stage ◽

Snare Complex ◽

Complex Activity ◽

Sm Proteins ◽

Complex Control ◽

Working Model ◽

Direct Penetration ◽

Snare Regulators

AbstractZippering of SNARE complexes spanning docked membranes is essential for most intracellular fusion events. Here we explore how SNARE regulators operate on discrete zippering states. The formation of a metastable trans-complex, catalyzed by HOPS and its SM subunit Vps33, is followed by subsequent zippering transitions that increase the probability of fusion. Operating independently of Sec18 catalysis, Sec17 either inhibits or stimulates SNARE-mediated fusion. If HOPS or Vps33 are absent, Sec17 inhibits fusion at an early stage. Thus, HOPS and Vps33 accelerate SNARE zippering, particularly in the presence of otherwise inhibitory Sec17. Once SNAREs are partially-zipped, Sec17 promotes fusion in either the presence or absence of HOPS — but with faster kinetics when HOPS is absent. Our data further indicate that Sec17 promotes fusion both through its direct penetration of the membrane and by enhancing C-terminal SNARE zippering. In a working model, the interplay among Sec17, Sec18, SMs, and SNARE zippering can explain why SM proteins are indispensable for SNARE-mediated fusion in vivo.Impact statementSec17 is shown to have divergent effects on pre-fusion SNARE complex activity, depending on the state of SNARE zippering. HOPS, an SM-tether complex, controls the outcome of Sec17-SNARE engagement. The results suggest a coherent working model for SM activity in vivo.

SM proteins Sly1 and Vps33 co-assemble with Sec17 and SNARE complexes to oppose SNARE disassembly by Sec18

eLife ◽

10.7554/elife.02272 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 50

Author(s):

Braden T Lobingier ◽

Daniel P Nickerson ◽

Sheng-Ying Lo ◽

Alexey J Merz

Keyword(s):

Snare Complex ◽

Wild Type ◽

Sm Proteins ◽

Working Model ◽

Conformational Rearrangements ◽

Kinetic Proofreading

Secretory and endolysosomal fusion events are driven by SNAREs and cofactors, including Sec17/α-SNAP, Sec18/NSF, and Sec1/Munc18 (SM) proteins. SMs are essential for fusion in vivo, but the basis of this requirement is enigmatic. We now report that, in addition to their established roles as fusion accelerators, SM proteins Sly1 and Vps33 directly shield SNARE complexes from Sec17- and Sec18-mediated disassembly. In vivo, wild-type Sly1 and Vps33 function are required to withstand overproduction of Sec17. In vitro, Sly1 and Vps33 impede SNARE complex disassembly by Sec18 and ATP. Unexpectedly, Sec17 directly promotes selective loading of Sly1 and Vps33 onto cognate SNARE complexes. A large thermodynamic barrier limits SM binding, implying that significant conformational rearrangements are involved. In a working model, Sec17 and SMs accelerate fusion mediated by cognate SNARE complexes and protect them from NSF-mediated disassembly, while mis-assembled or non-cognate SNARE complexes are eliminated through kinetic proofreading by Sec18.

CSPα reduces aggregates and rescues striatal dopamine release in αsynuclein transgenic mice

10.1101/2020.07.31.229153 ◽

2020 ◽

Author(s):

L Caló ◽

E Hidari ◽

M Wegrzynowicz ◽

JW Dalley ◽

BL Schneider ◽

...

Keyword(s):

Dopamine Release ◽

Early Stage ◽

Synaptic Function ◽

Snare Complex ◽

Early Event ◽

Vesicle Recycling ◽

Cysteine String Protein ◽

And Function

AbstractαSynuclein aggregation at the synapse is an early event in Parkinson’s disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and αsynuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like αsynuclein, chaperones the SNARE complex assembly and neurotransmitter release. αSynuclein can rescue neurodegeneration in CSPαKO mice. However, whether αsynuclein aggregation alters CSPα expression and function is unknown. Here we show that αsynuclein aggregation at the synapse induces a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with αsynuclein aggregates and in vivo reduces synaptic αsynuclein aggregates restoring normal dopamine release in 1-120hαsyn mice. These novel findings reveal a mechanism by which αsynuclein aggregation alters CSPα at the synapse, and show that CSPα rescues αsynuclein aggregation-related phenotype in 1-120hαsyn mice similar to the effect of αsynuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage PD.

Binding of UNC-18 to the N-terminus of syntaxin is essential for neurotransmission in Caenorhabditis elegans

Biochemical Journal ◽

10.1042/bj20081956 ◽

2009 ◽

Vol 418 (1) ◽

pp. 73-80 ◽

Cited By ~ 43

Author(s):

James R. Johnson ◽

Pawel Ferdek ◽

Lu-Yun Lian ◽

Jeff W. Barclay ◽

Robert D. Burgoyne ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Membrane Fusion ◽

Snare Complex ◽

Binding Modes ◽

Sm Proteins ◽

Closed Conformation ◽

Physiological Relevance ◽

N Terminus

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) are widely accepted to drive all intracellular membrane fusion events. SM (Sec1/Munc18-like) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least three modes: (i) to a closed conformation of syntaxin; (ii) to the syntaxin N-terminus; and (iii) to the assembled SNARE complex. Munc18-1 exhibits all three binding modes and recent in vitro reconstitution assays suggest that its interaction with the syntaxin N-terminus is essential for neuronal SNARE complex binding and efficient membrane fusion. To investigate the physiological relevance of these binding modes, we studied the UNC-18/UNC-64 SM/SNARE pair, which is essential for neuronal exocytosis in Caenorhabditis elegans. Mutations in the N-terminus of UNC-64 strongly inhibited binding to UNC-18, as did mutations targeting closed conformation binding. Complementary mutations in UNC-18 designed to selectively impair binding to either closed syntaxin or its N-terminus produced a similarly strong inhibition of UNC-64 binding. Therefore high-affinity UNC18/UNC-64 interaction in vitro involves both binding modes. To determine the physiological relevance of each mode, unc-18-null mutant worms were transformed with wild-type or mutant unc-18 constructs. The UNC-18(R39C) construct, that is defective in closed syntaxin binding, fully rescued the locomotion defects of the unc-18 mutant. In contrast, the UNC-18(F113R) construct, that is defective in binding to the N-terminus of UNC-64, provided no rescue. These results suggest that binding of UNC-18 to closed syntaxin is dispensable for membrane fusion, whereas interaction with the syntaxin N-terminus is essential for neuronal exocytosis in vivo.

Structure-based Functional Analysis Reveals a Role for the SM Protein Sly1p in Retrograde Transport to the Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0114 ◽

2005 ◽

Vol 16 (9) ◽

pp. 3951-3962 ◽

Cited By ~ 16

Author(s):

Yujie Li ◽

Dieter Gallwitz ◽

Renwang Peng

Keyword(s):

Endoplasmic Reticulum ◽

Mutational Analysis ◽

Retrograde Transport ◽

Snare Complex ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Sm Proteins ◽

Sm Protein

Sec1p/Munc18 (SM) proteins are essential for membrane fusion events in eukaryotic cells. Here we describe a systematic, structure-based mutational analysis of the yeast SM protein Sly1p, which was previously shown to function in anterograde endoplasmic reticulum (ER)-to-Golgi and intra-Golgi protein transport. Five new temperature-sensitive (ts) mutants, each carrying a single amino acid substitution in Sly1p, were identified. Unexpectedly, not all of the ts mutants exhibited striking anterograde ER-to-Golgi transport defects. For example, in cells of the novel sly1-5 mutant, transport of newly synthesized lysosomal and secreted proteins was still efficient, but the ER-resident Kar2p/BiP was missorted to the outside of the cell, and two proteins, Sed5p and Rer1p, which normally shuttle between the Golgi and the ER, failed to relocate to the ER. We also discovered that in vivo, Sly1p was associated with a SNARE complex formed on the ER, and that in vitro, the SM protein directly interacted with the ER-localized nonsyntaxin SNAREs Use1p/Slt1p and Sec20p. Furthermore, several conditional mutants defective in Golgi-to-ER transport were synthetically lethal with sly1-5. Together, these results indicate a previously unrecognized function of Sly1p in retrograde transport to the endoplasmic reticulum.

Sec17 (α-SNAP) and an SM-tethering complex regulate the outcome of SNARE zippering in vitro and in vivo

eLife ◽

10.7554/elife.27396 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 16

Author(s):

Matthew L Schwartz ◽

Daniel P Nickerson ◽

Braden T Lobingier ◽

Rachael L Plemel ◽

Mengtong Duan ◽

...

Keyword(s):

Early Stage ◽

Rate Limiting Step ◽

Limiting Step ◽

Trans Complex ◽

Snare Regulators ◽

Rate Limiting

Zippering of SNARE complexes spanning docked membranes is essential for most intracellular fusion events. Here, we explore how SNARE regulators operate on discrete zippering states. The formation of a metastable trans-complex, catalyzed by HOPS and its SM subunit Vps33, is followed by subsequent zippering transitions that increase the probability of fusion. Operating independently of Sec18 (NSF) catalysis, Sec17 (α-SNAP) either inhibits or stimulates SNARE-mediated fusion. If HOPS or Vps33 are absent, Sec17 inhibits fusion at an early stage. Thus, Vps33/HOPS promotes productive SNARE assembly in the presence of otherwise inhibitory Sec17. Once SNAREs are partially zipped, Sec17 promotes fusion in either the presence or absence of HOPS, but with faster kinetics when HOPS is absent, suggesting that ejection of the SM is a rate-limiting step.

CSPα reduces aggregates and rescues striatal dopamine release in αsynuclein transgenic mice

Brain ◽

10.1093/brain/awab076 ◽

2021 ◽

Author(s):

Laura Caló ◽

Eric Hidari ◽

Michal Wegrzynowicz ◽

Jeffrey W Dalley ◽

Bernard L Schneider ◽

...

Keyword(s):

Dopamine Release ◽

Early Stage ◽

Synaptic Function ◽

Snare Complex ◽

Early Event ◽

Vesicle Recycling ◽

Cysteine String Protein ◽

And Function

Abstract αSynuclein aggregation at the synapse is an early event in Parkinson’s disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and αsynuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like αsynuclein, chaperones the SNARE complex assembly and controls neurotransmitter release. αSynuclein can rescue neurodegeneration in CSPαKO mice. However, whether αsynuclein aggregation alters CSPα expression and function is unknown. Here we show that αsynuclein aggregation at the synapse is associated with a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with αsynuclein aggregates and in vivo reduces synaptic αsynuclein aggregates and increasing monomeric asynuclein, restoring normal dopamine release in 1–120hαSyn mice. These novel findings reveal a mechanism by which αsynuclein aggregation alters CSPα at the synapse, and show that CSPα rescues αsynuclein aggregation-related phenotype in 1-120hαSyn mice similar to the effect of αsynuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage PD.

Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

Molecular and Cellular Biology ◽

10.1128/mcb.00857-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4641-4651 ◽

Cited By ~ 40

Author(s):

Junjiang Fu ◽

Ho-Geun Yoon ◽

Jun Qin ◽

Jiemin Wong

Keyword(s):

Rna Polymerase Ii ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Elongation Complex ◽

Complex Activity ◽

Interacting Protein ◽

Pol Ii ◽

Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

Upregulations of Clcn3 and P-Gp Provoked by Lens Osmotic Expansion in Rat Galactosemic Cataract

Journal of Diabetes Research ◽

10.1155/2017/3472735 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8

Author(s):

Lixia Ji ◽

Lixia Cheng ◽

Zhihong Yang

Keyword(s):

Osmotic Stress ◽

Early Stage ◽

Lens Epithelial Cells ◽

Anterior Capsule ◽

Glycoprotein P ◽

Protein Levels ◽

P Glycoprotein ◽

Sugar Cataract

Objective.Lens osmotic expansion, provoked by overactivated aldose reductase (AR), is the most essential event of sugar cataract. Chloride channel 3 (Clcn3) is a volume-sensitive channel, mainly participating in the regulation of cell fundamental volume, and P-glycoprotein (P-gp) acts as its modulator. We aim to study whether P-gp and Clcn3 are involved in lens osmotic expansion of galactosemic cataract.Methods and Results.In vitro, lens epithelial cells (LECs) were primarily cultured in gradient galactose medium (10–60 mM), more and more vacuoles appeared in LEC cytoplasm, and mRNA and protein levels of AR, P-gp, and Clcn3 were synchronously upregulated along with the increase of galactose concentration. In vivo, we focused on the early stage of rat galactosemic cataract, amount of vacuoles arose from equatorial area and scattered to the whole anterior capsule of lenses from the 3rd day to the 9th day, and mRNA and protein levels of P-gp and Clcn3 reached the peak around the 9th or 12th day.Conclusion. Galactosemia caused the osmotic stress in lenses; it also markedly leads to the upregulations of AR, P-gp, and Clcn3 in LECs, together resulting in obvious osmotic expansion in vitro and in vivo.

Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

The Journal of Cell Biology ◽

10.1083/jcb.200206113 ◽

2002 ◽

Vol 159 (6) ◽

pp. 993-1004 ◽

Cited By ~ 126

Author(s):

Christine L. Humphries ◽

Heath I. Balcer ◽

Jessica L. D'Agostino ◽

Barbara Winsor ◽

David G. Drubin ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Complex Activity ◽

Actin Binding Protein ◽

Coiled Coil Domain ◽

Allele Specific ◽

And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

Rationally Designing Simple Rod-Like Amphiphilic NIR-Emissive AIE Probes for Precise In-Vivo Detection of Aβ Fibrils/Plaques at A Super-Early Stage

10.26434/chemrxiv.13699222.v1 ◽

2021 ◽

Author(s):

Yipu Wang ◽

Dong Mei ◽

Xinyi Zhang ◽

Da-Hui Qu ◽

Ju Mei ◽

...

Keyword(s):

High Performance ◽

Ex Vivo ◽

Early Stage ◽

Signal To Noise Ratio ◽

High Signal ◽

In Vivo Detection ◽

Different Strains ◽

Aβ Fibrils

With increase of social aging, Alzheimer's disease (AD) has been one of the serious diseases threatening human health. The occurrence of Aβ fibrils or plaques is recognized as the hallmark of AD. Currently, optical imaging has stood out to be a promising technique for the imaging of Aβ fibrils/plaques and the diagnosis of AD. However, restricted by their poor blood-brain barrier (BBB) penetrability, short-wavelength excitation and emission, and aggregation-caused quenching (ACQ) effect, the clinically used gold-standard optical probes such as <a>thioflavin</a> T (ThT) and thioflavin S (ThS), are not effective enough in the early diagnosis of AD in vivo. Herein, we put forward an “all-in-one” design principle and demonstrate its feasibility in developing high-performance fluorescent probes which are specific to Aβ fibrils/plaques and promising for super-early in-vivo diagnosis of AD. As a proof of concept, a simple rod-like amphiphilic NIR fluorescent AIEgen, i.e., AIE-CNPy-AD, is developed by taking the specificity, BBB penetration ability, deep-tissue penetration capacity, high signal-to-noise ratio (SNR) into consideration. AIE-CNPy-AD is constituted by connecting the electron-donating and accepting moieties through single bonds and tagging with a propanesulfonate tail, giving rise to the NIR fluorescence, aggregation-induced emission (AIE) effect, amphiphilicity, and rod-like structure, which in turn result in high binding-affinity and excellent specificity to Aβ fibrils/plaques, satisfactory ability to penetrate BBB and deep tissues, ultrahigh SNR and sensitivity, and high-fidelity imaging capability. In-vitro, ex-vivo, and in-vivo <a>identifying of Aβ fibrils/plaques</a> in different strains of mice indicate that AIE-CNPy-AD holds the universality to the detection of Aβ fibrils/plaques. It is noteworthy that AIE-CNPy-AD is even able to trace the small and sparsely distributed Aβ fibrils/plaques in very young AD model mice such as 4-month-old APP/PS1 mice which are reported to be the youngest mice to have Aβ deposits in brains, suggesting its great potential in diagnosis and intervention of AD at a super-early stage.