Phosphatidylinositol (4, 5)‐bisphosphate targets double C2 domain protein B to the plasma membrane

Michaeli, Lirin; Gottfried, Irit; Bykhovskaia, Maria; Ashery, Uri

doi:10.1111/tra.12528

Cited by 16 publications

(19 citation statements)

References 70 publications

(141 reference statements)

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“…Vesicle fusion promotes the integration of GLUT4 into the PM, which facilitates glucose uptake. Several reports show that DOC2B is a soluble protein and can exist in the cytosol, consistent with DOC2B having no transmembrane domain [4,18,19]. DOC2B is speculated to regulate insulin-stimulated GLUT4 translocation via insulininduced DOC2B translocation from the cytosol to the PM, where the target-associated SNARE (t-SNARE) proteins reside [4,18,20,21].…”

Section: Introductionmentioning

confidence: 85%

“…Several reports show that DOC2B is a soluble protein and can exist in the cytosol, consistent with DOC2B having no transmembrane domain [4,18,19]. DOC2B is speculated to regulate insulin-stimulated GLUT4 translocation via insulininduced DOC2B translocation from the cytosol to the PM, where the target-associated SNARE (t-SNARE) proteins reside [4,18,20,21]. At the PM, insulin stimulation increases DOC2B binding to phosphorylated syntaxin binding protein (STXBP)3, increasing SNARE complex formation [9,11,22].…”

Section: Introductionmentioning

confidence: 88%

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DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

Zhang

Merz

et al. 2019

Diabetologia

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Aims/hypothesis Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain β (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. Methods Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, coimmunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B. Results We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM. Conclusions/interpretation These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes. Karla E. Merz and Arianne Aslamy contributed equally to this work.Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00125-019-4824-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

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Section: Introductionmentioning

confidence: 85%

Section: Introductionmentioning

confidence: 88%

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

Zhang

Merz

et al. 2019

Diabetologia

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“…These results agree with quantitative thermodynamic studies [64], which observed the Ca 2+ -induced membrane penetration experimentally. Notably, a similar configuration for the attachment of the C2B module to PIP2 was demonstrated for the Syt1 analog DOC2B [67], suggesting a general mechanism for the membrane anchoring of C2B domains.…”

Section: Discussionmentioning

confidence: 55%

SNARE Complex Alters the Interactions of the Ca²⁺sensor Synaptotagmin 1 with Lipid Bilayers

Bykhovskaia

2020

Preprint

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Neuronal transmitters are packed in synaptic vesicles (SVs) and released by fusion of SVs with the presynaptic membrane (PM). SVs are attached to PM by the SNARE protein complex, and fusion is triggered by the Ca 2+ sensor Synaptotagmin 1 (Syt1). Although Syt1 and SNARE proteins have been extensively studied, it is not yet fully understood how the interactions of Syt1 with lipids and the SNARE complex induce fusion. To address this fundamental problem, we took advantage of Anton2 supercomputer, a unique computational environment, which enables simulating the dynamics of molecular systems at a scale of microseconds. Our simulations produced a dynamic all-atom model of the prefusion protein-lipid complex and demonstrated in silico how the Syt1-SNARE complex triggers fusion. AbstractRelease of neuronal transmitters from nerve terminals is triggered by the molecular Ca 2+ sensor Synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca 2+ binding. Prior to fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which binds the C2B domain of Syt1. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. The MD simulations showed that the C2AB tandem of Syt1 can either bridge SV and PM or immerse into PM, and that the latter configuration is more favorable energetically. Surprisingly, C2 domains did not cooperate in penetrating into PM, but instead mutually hindered the lipid penetration. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the Syt1-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains of Syt1 and promotes the immersion of both domains into the PM bilayer. Subsequently, we simulated this pre-fusion protein complex between lipid bilayers imitating PM and SV and found that the immersion of Syt1 into the PM bilayer within this complex promotes PM curvature and leads to lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex. 4The palmitoyl oleyl phosphatidylcholine (POPC) lipid bilayers were generated using VMD. The initial structure of anionic lipid bilayer containing phosphatidylserine (POPS) and PIP2 , POPC:POPS:PIP2 (75:20:5) [74] was kindly provided by Dr. J. Wereszczynski (Illinois Institute of Technology). In all the systems, the lipid bilayers were positioned in the XY plain.The initial structures for the isolated C2A [75]) and C2B [76] domains in their Ca 2+ -bound forms were obtained from crystallography studies (1BYN and 1TJX, respectively in the Protein Data Base). The initial structures of the isol...

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“…In cell-free assays, Doc2b interacts with the SNARE complex to promote fusion of SNARE-liposomes (Groffen et al, 2010; Yao et al, 2011). Doc2b C 2 domains bind to phosphatidylserine-containing membranes in a Ca 2+ -dependent manner (Groffen et al, 2010) but also to PI(4,5)P 2 , a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane (Michaeli et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…However, alteration of this interaction by mutations in the MID domain have no effect on Ca 2+ -induced Doc2b migration to the membrane (Groffen et al, 2004; Gaffaney et al, 2014). The presence of PI(4,5)P 2 targets Doc2b to the plasma membrane upon [Ca 2+ ] i elevation (Michaeli et al, 2017). Hence, Doc2b could support exocytosis by both of several mechanisms: i) together with Munc-13 for vesicle priming or superpriming; ii) Ca 2+ -dependently by enhancing membrane fusion.…”

Section: Introductionmentioning

confidence: 99%

Doc2b Ca²⁺-binding site mutants act as a gain of function at rest and loss of function during neuronal activity

Bourgeois-Jaarsma

Verhage

Groffen

2019

Preprint

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21Communication between neurons involves presynaptic neurotransmitter release which 22 can be evoked by action potentials or occur spontaneously as a result of stochastic 23 vesicle fusion. The Ca 2+ -binding double C2 proteins Doc2a and -b regulate both 24 spontaneous and asynchronous evoked release, but the mechanism remains unclear. 25Here, we compared wildtype Doc2b with two Ca 2+ binding site mutants named DN and 26 6A, respectively considered gain-and loss-of function mutants and carrying the 27 substitutions D218,220N or D163,218,220,303,357,359A. We found that both mutants 28 bound phospholipids at low free Ca 2+ concentrations and were membrane-associated 29 in neurons at rest, mimicking a Ca 2+ activated state. Their overexpression in 30 hippocampal primary neurons culture had similar effects on spontaneous and evoked 31 release, inducing higher mEPSC frequencies and increased short-term depression. 32Together, these data suggest that the DN and 6A mutants both act as gain-of-function 33 mutants at resting conditions but as loss-of-function during neuronal activity. 34 35 36

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Phosphatidylinositol (4, 5)‐bisphosphate targets double C2 domain protein B to the plasma membrane

Cited by 16 publications

References 70 publications

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

SNARE Complex Alters the Interactions of the Ca²⁺sensor Synaptotagmin 1 with Lipid Bilayers

Doc2b Ca²⁺-binding site mutants act as a gain of function at rest and loss of function during neuronal activity

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Phosphatidylinositol (4, 5)‐bisphosphate targets double C2 domain protein B to the plasma membrane

Cited by 16 publications

References 70 publications

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

SNARE Complex Alters the Interactions of the Ca2+sensor Synaptotagmin 1 with Lipid Bilayers

Doc2b Ca2+-binding site mutants act as a gain of function at rest and loss of function during neuronal activity

Contact Info

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About

SNARE Complex Alters the Interactions of the Ca²⁺sensor Synaptotagmin 1 with Lipid Bilayers

Doc2b Ca²⁺-binding site mutants act as a gain of function at rest and loss of function during neuronal activity