Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

Rathore, Shailendra; Liu, Yinghui; Yu, Haijia; Wan, Chunrong; Lee, Myeongseon; Yin, Qian; Stowell, Michael H. B.; Shen, Jingshi

doi:10.1016/j.celrep.2019.11.107

Cited by 25 publications

(14 citation statements)

References 93 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The leakage assay was conducted in a 96-well microplate at 37°C, and sulforhodamine B fluorescence (ex565/em585 nm) was measured every 2 minutes in a BioTek Synergy H1 plate reader. Ten μL of 10% CHAPSO (Sigma-Aldrich) was added to each sample at the end of the reaction [ 123 ]. Leakage data were presented as the percentage of maximum fluorescence change based on three biological replicates.…”

Section: Methodsmentioning

confidence: 99%

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids

et al. 2020

Self Cite

View full text Add to dashboard Cite

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.

show abstract

Section: Methodsmentioning

confidence: 99%

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…The interactions of soluble factors with liposomes were measured using a liposome coflotation assay 60,71,77,80 . Soluble proteins were incubated with liposomes at 4°C with gentle agitation.…”

Section: Methodsmentioning

confidence: 99%

“…The interactions of soluble factors with liposomes were measured using a liposome coflotation assay. 60,71,77,80…”

Section: Liposome Coflotation Assaymentioning

confidence: 99%

Genetic evidence for an inhibitory role of tomosyn in insulin‐stimulated GLUT4 exocytosis

Wang

Liu

Crisman

et al. 2020

Traffic

Self Cite

View full text Add to dashboard Cite

Exocytosis is a vesicle fusion process driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A classic exocytic pathway is insulinstimulated translocation of the glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane in adipocytes and skeletal muscles. The GLUT4 exocytic pathway plays a central role in maintaining blood glucose homeostasis and is compromised in insulin resistance and type 2 diabetes. A candidate regulator of GLUT4 exocytosis is tomosyn, a soluble protein expressed in adipocytes. Tomosyn directly binds to GLUT4 exocytic SNAREs in vitro but its role in GLUT4 exocytosis was unknown. In this work, we used CRISPR-Cas9 genome editing to delete the two tomosyn-encoding genes in adipocytes. We observed that both basal and insulinstimulated GLUT4 exocytosis was markedly elevated in the double knockout (DKO) cells. By contrast, adipocyte differentiation and insulin signaling remained intact in the DKO adipocytes. In a reconstituted liposome fusion assay, tomosyn inhibited all the SNARE complexes underlying GLUT4 exocytosis. The inhibitory activity of tomosyn was relieved by NSF and α-SNAP, which act in concert to remove tomosyn from GLUT4 exocytic SNAREs. Together, these studies revealed an inhibitory role for tomosyn in insulin-stimulated GLUT4 exocytosis in adipocytes. We suggest that tomosyn-arrested SNAREs represent a reservoir of fusion capacity that could be harnessed to treat patients with insulin resistance and type 2 diabetes.

show abstract

“…We suggest that Munc18-1 and Munc13-1 highly regulate the whole process of SNARE complex assembly in evoked fusion, whereas their predominant roles in spontaneous fusion lie in vesicle docking and membrane association prior to SNARE assembly. Increasing evidence has indicated that the JLR of synaptobrevin-2 serves as an important element not only for force transmission but also for regulations by multiple fusion components such as calmodulin, Munc13s, and phospholipids (Quetglas et al, 2000 ; Kweon et al, 2003 ; Bowen and Brunger, 2006 ; Ellena et al, 2009 ; Brewer et al, 2011 ; Borisovska et al, 2012 ; Han et al, 2016 ; Rathore et al, 2019 ; Wang et al, 2019 ). Although length-increasing insertions in the synaptobrevin-2 JLR do not disturb Munc13-mediated N-terminal SNARE assembly ( Supplementary Figure 5 ), structural perturbations in this region may affect cooperative interplay among the JLR, Munc13s, and phospholipids, therefore altering fusion competence.…”

Section: Discussionmentioning

confidence: 99%

Structural Roles for the Juxtamembrane Linker Region and Transmembrane Region of Synaptobrevin 2 in Membrane Fusion

Zhu

2021

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

Formation of the trans-SNARE complex is believed to generate a force transfer to the membranes to promote membrane fusion, but the underlying mechanism remains elusive. In this study, we show that helix-breaking and/or length-increasing insertions in the juxtamembrane linker region of synaptobrevin-2 exert diverse effects on liposome fusion, in a manner dependent on the insertion position relative to the two conserved tryptophan residues (W89/W90). Helical extension of synaptobrevin-2 to W89/W90 is a prerequisite for initiating membrane merger. The transmembrane region of synaptobrevin-2 enables proper localization of W89/W90 at the membrane interface to gate force transfer. Besides, our data indicate that the SNARE regulatory components Munc18-1 and Munc13-1 impose liposome fusion strong demand on tight coupling between the SNARE motif and the transmembrane region of synaptobrevin-2.

show abstract

Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

Cited by 25 publications

References 93 publications

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids

Genetic evidence for an inhibitory role of tomosyn in insulin‐stimulated GLUT4 exocytosis

Structural Roles for the Juxtamembrane Linker Region and Transmembrane Region of Synaptobrevin 2 in Membrane Fusion

Contact Info

Product

Resources

About