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Thomas Weimbs - One of the best experts on this subject based on the ideXlab platform.
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The Habc domain of Syntaxin 3 is a ubiquitin binding domain.
Scientific reports, 2020Co-Authors: Adrian J. Giovannone, Elena Reales, Pallavi Bhattaram, Sirpi Nackeeran, Adam B. Monahan, Rashid Syed, Thomas WeimbsAbstract:Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members. The Syntaxin Habc domain has previously been found to be structurally very similar to the GAT domain present in GGA family members and related proteins that are otherwise completely unrelated to Syntaxins. Because the GAT domain has been found to be a ubiquitin binding domain we hypothesized that the Habc domain of Syntaxins may also bind to ubiquitin. Here, we report that the Habc domain of Syntaxin 3 (Stx3) indeed binds to monomeric ubiquitin with low affinity. This domain binds efficiently to K63-linked poly-ubiquitin chains within a narrow range of chain lengths but not to K48-linked poly-ubiquitin chains. Other Syntaxin family members also bind to K63-linked poly-ubiquitin chains but with different chain length specificities. Molecular modeling suggests that residues of the GGA3-GAT domain known to be important for ionic and hydrophobic interactions with ubiquitin may have equivalent, conserved residues within the Habc domain of Stx3. We conclude that the Syntaxin Habc domain and the GAT domain are both structurally and functionally related, and likely share a common ancestry despite sequence divergence. Binding of Ubiquitin to the Habc domain may regulate the function of Syntaxins in membrane fusion or may suggest additional functions of this protein family.
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soluble Syntaxin 3 functions as a transcriptional regulator
Journal of Biological Chemistry, 2018Co-Authors: Adrian J. Giovannone, Christine Winterstein, Elena Reales, Pallavi Bhattaram, Seng Hui Low, Julie E Baggs, Matthew A Lalli, John B Hogenesch, Thomas WeimbsAbstract:Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Stx3 (Syntaxin 3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform that we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5 (RAN-binding protein 5), targets to the nucleus, and interacts physically and functionally with several transcription factors, including ETV4 (ETS variant 4) and ATF2 (activating transcription factor 2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other Syntaxins were identified, suggesting that nuclear signaling is a conserved, novel function common among these membrane-trafficking proteins.
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soluble Syntaxin 3 functions as a transcription regulator
bioRxiv, 2017Co-Authors: Adrian J. Giovannone, Christine Winterstein, Elena Reales, Pallavi Bhattaram, Seng Hui Low, Julie E Baggs, Matthew A Lalli, John B Hogenesch, Thomas WeimbsAbstract:Syntaxins - a conserved family of SNARE proteins - contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Syntaxin 3 (Stx3) unexpectedly also functions as a nuclear regulator of gene expression. Alternative splicing leads to a soluble isoform, termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5, targets to the nucleus and interacts physically and functionally with several transcription factors, including ETV4 and ATF2. Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer vs. normal breast tissue. Inhibition of endogenous Stx3S expression leads to changes in the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other Syntaxins were identified suggesting that nuclear signaling is a conserved, novel function common among these membrane trafficking proteins.
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monoubiquitination of Syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes
Molecular Biology of the Cell, 2017Co-Authors: Adrian J. Giovannone, Elena Reales, Pallavi Bhattaram, Alberto Fraileramos, Thomas WeimbsAbstract:Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral-but not the apical-plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion.
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mono ubiquitination of Syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes
bioRxiv, 2017Co-Authors: Adrian J. Giovannone, Elena Reales, Pallavi Bhattaram, Alberto Fraileramos, Thomas WeimbsAbstract:Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes though how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes mono-ubiquitination in a conserved polybasic domain. Stx3 present at the basolateral, but not the apical, plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs) and excreted in exosomes. A non-ubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploit this pathway for virion excretion.
Frederic Calon - One of the best experts on this subject based on the ideXlab platform.
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high fat diet aggravates amyloid beta and tau pathologies in the 3xtg ad mouse model
Neurobiology of Aging, 2010Co-Authors: Carl Julien, Cyntia Tremblay, Alix Phivilay, Line Berthiaume, Vincent Emond, Pierre Julien, Frederic CalonAbstract:Abstract To investigate potential dietary risk factors of Alzheimer’s disease (AD), triple transgenic (3xTg-AD) mice were exposed from 4 to 13 months of age to diets with a low n-3:n-6 polyunsaturated fatty acid (PUFA) ratio incorporated in either low-fat (5% w/w) or high-fat (35% w/w) formulas and compared with a control diet. The n-3:n-6 PUFA ratio was decreased independently of the dietary treatments in the frontal cortex of 3xTg-AD mice compared to non-transgenic littermates. Consumption of a high-fat diet with a low n-3:n-6 PUFA ratio increased amyloid-β (Aβ) 40 and 42 concentrations in detergent-insoluble extracts of parieto-temporal cortex homogenates from 3xTg-AD mice. Low n-3:n-6 PUFA intake ratio increased insoluble tau regardless of total fat consumption, whereas high-fat diet incorporating a low n-3:n-6 PUFA ratio also increased soluble tau compared to controls. Moreover, the high-fat diet decreased cortical levels of the postsynaptic marker drebrin, while leaving presynaptic proteins synaptophysin, SNAP-25 and Syntaxin 3 unchanged. Overall, these results suggest that high-fat consumption combined with low n-3 PUFA intake promote AD-like neuropathology.
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high fat diet aggravates amyloid beta and tau pathologies in the 3xtg ad mouse model
Neurobiology of Aging, 2010Co-Authors: Carl Julien, Cyntia Tremblay, Alix Phivilay, Line Berthiaume, Vincent Emond, Pierre Julien, Frederic CalonAbstract:To investigate potential dietary risk factors of Alzheimer's disease (AD), triple transgenic (3xTg-AD) mice were exposed from 4 to 13 months of age to diets with a low n-3:n-6 polyunsaturated fatty acid (PUFA) ratio incorporated in either low-fat (5% w/w) or high-fat (35% w/w) formulas and compared with a control diet. The n-3:n-6 PUFA ratio was decreased independently of the dietary treatments in the frontal cortex of 3xTg-AD mice compared to non-transgenic littermates. Consumption of a high-fat diet with a low n-3:n-6 PUFA ratio increased amyloid-beta (Abeta) 40 and 42 concentrations in detergent-insoluble extracts of parieto-temporal cortex homogenates from 3xTg-AD mice. Low n-3:n-6 PUFA intake ratio increased insoluble tau regardless of total fat consumption, whereas high-fat diet incorporating a low n-3:n-6 PUFA ratio also increased soluble tau compared to controls. Moreover, the high-fat diet decreased cortical levels of the postsynaptic marker drebrin, while leaving presynaptic proteins synaptophysin, SNAP-25 and Syntaxin 3 unchanged. Overall, these results suggest that high-fat consumption combined with low n-3 PUFA intake promote AD-like neuropathology.
Shuzo Sugita - One of the best experts on this subject based on the ideXlab platform.
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munc18b increases insulin granule fusion restoring deficient insulin secretion in type 2 diabetes human and goto kakizaki rat islets with improvement in glucose homeostasis
EBioMedicine, 2017Co-Authors: Tairan Qin, Subhankar Dolai, Li Xie, Dan Zhu, Tao Liang, Youhou Kang, Shuzo Sugita, Noriko Takahashi, Claesgoran OstensonAbstract:Reduced pancreatic islet levels of Munc18a/SNARE complex proteins have been postulated to contribute to the deficient glucose-stimulated insulin secretion (GSIS) in type-2 diabetes (T2D). Whereas much previous work has purported Munc18a/SNARE complex (Syntaxin-1A/VAMP-2/SNAP25) to be primarily involved in predocked secretory granule (SG) fusion, less is known about newcomer SGs that undergo minimal docking time at the plasma membrane before fusion. Newcomer SG fusion has been postulated to involve a distinct SM/SNARE complex (Munc18b/Syntaxin-3/VAMP8/SNAP25), whose levels we find also reduced in islets of T2D humans and T2D Goto-Kakizaki (GK) rats. Munc18b overexpression by adenovirus infection (Ad-Munc18b), by increasing assembly of Munc18b/SNARE complexes, mediated increased fusion of not only newcomer SGs but also predocked SGs in T2D human and GK rat islets, resulting in rescue of the deficient biphasic GSIS. Infusion of Ad-Munc18b into GK rat pancreas led to sustained improvement in glucose homeostasis. However, Munc18b overexpression in normal islets increased only newcomer SG fusion. Therefore, Munc18b could potentially be deployed in human T2D to rescue the deficient GSIS.
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chaperoning of closed Syntaxin 3 through lys46 and glu59 in domain 1 of munc18 proteins is indispensable for mast cell exocytosis
Journal of Cell Science, 2015Co-Authors: Naryum Bin, Dan Zhu, Chang Hun Jung, Byungjin Kim, Prashanth Chandrasegram, Ekaterina Turlova, Herbert Y Gaisano, Hongshuo Sun, Shuzo SugitaAbstract:Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of Syntaxin isoforms. However, the key Syntaxin that, crucially, participates in the degranulation – whose levels and intracellular localization are regulated by Munc18-2 – remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of Syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' Syntaxin-3. Furthermore, knockdown of Syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed Syntaxin-3, which is essential for degranulation by chaperoning Syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.
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Syntaxin 3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells
Diabetologia, 2013Co-Authors: Dan Zhu, Huanli Xie, Youhou Kang, Shuzo Sugita, E Koo, Edwin P Kwan, Seungmee Park, Herbert Y GaisanoAbstract:Aims/hypothesis The molecular basis of the exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells remains unclear. Syntaxin (SYN)-1A and SYN-4 have been shown to mediate insulin exocytosis. The insulin-secretory function of SYN-3, which is particularly abundant in SGs, is unclear.
Carl Julien - One of the best experts on this subject based on the ideXlab platform.
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high fat diet aggravates amyloid beta and tau pathologies in the 3xtg ad mouse model
Neurobiology of Aging, 2010Co-Authors: Carl Julien, Cyntia Tremblay, Alix Phivilay, Line Berthiaume, Vincent Emond, Pierre Julien, Frederic CalonAbstract:Abstract To investigate potential dietary risk factors of Alzheimer’s disease (AD), triple transgenic (3xTg-AD) mice were exposed from 4 to 13 months of age to diets with a low n-3:n-6 polyunsaturated fatty acid (PUFA) ratio incorporated in either low-fat (5% w/w) or high-fat (35% w/w) formulas and compared with a control diet. The n-3:n-6 PUFA ratio was decreased independently of the dietary treatments in the frontal cortex of 3xTg-AD mice compared to non-transgenic littermates. Consumption of a high-fat diet with a low n-3:n-6 PUFA ratio increased amyloid-β (Aβ) 40 and 42 concentrations in detergent-insoluble extracts of parieto-temporal cortex homogenates from 3xTg-AD mice. Low n-3:n-6 PUFA intake ratio increased insoluble tau regardless of total fat consumption, whereas high-fat diet incorporating a low n-3:n-6 PUFA ratio also increased soluble tau compared to controls. Moreover, the high-fat diet decreased cortical levels of the postsynaptic marker drebrin, while leaving presynaptic proteins synaptophysin, SNAP-25 and Syntaxin 3 unchanged. Overall, these results suggest that high-fat consumption combined with low n-3 PUFA intake promote AD-like neuropathology.
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high fat diet aggravates amyloid beta and tau pathologies in the 3xtg ad mouse model
Neurobiology of Aging, 2010Co-Authors: Carl Julien, Cyntia Tremblay, Alix Phivilay, Line Berthiaume, Vincent Emond, Pierre Julien, Frederic CalonAbstract:To investigate potential dietary risk factors of Alzheimer's disease (AD), triple transgenic (3xTg-AD) mice were exposed from 4 to 13 months of age to diets with a low n-3:n-6 polyunsaturated fatty acid (PUFA) ratio incorporated in either low-fat (5% w/w) or high-fat (35% w/w) formulas and compared with a control diet. The n-3:n-6 PUFA ratio was decreased independently of the dietary treatments in the frontal cortex of 3xTg-AD mice compared to non-transgenic littermates. Consumption of a high-fat diet with a low n-3:n-6 PUFA ratio increased amyloid-beta (Abeta) 40 and 42 concentrations in detergent-insoluble extracts of parieto-temporal cortex homogenates from 3xTg-AD mice. Low n-3:n-6 PUFA intake ratio increased insoluble tau regardless of total fat consumption, whereas high-fat diet incorporating a low n-3:n-6 PUFA ratio also increased soluble tau compared to controls. Moreover, the high-fat diet decreased cortical levels of the postsynaptic marker drebrin, while leaving presynaptic proteins synaptophysin, SNAP-25 and Syntaxin 3 unchanged. Overall, these results suggest that high-fat consumption combined with low n-3 PUFA intake promote AD-like neuropathology.
Vesa M. Olkkonen - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the Munc18b-Syntaxin binding interface. Use of a mutant Munc18b to dissect the functions of Syntaxins 2 and 3.
The Journal of biological chemistry, 2002Co-Authors: Maria Kauppi, Gerd Wohlfahrt, Vesa M. OlkkonenAbstract:Abstract Munc18b is a mammalian Sec1-related protein that is abundant in epithelial cells and regulates vesicle transport to the apical plasma membrane. We constructed a homology model of Munc18b in complex with Syntaxin 3 based on the crystal structure of the neuronal Sec1·Syntaxin 1A complex. In this model we identified all residues in the interface between the two proteins that contribute directly to the interaction and mutagenized residues in Munc18b to alter its binding to Syntaxins 1A, 2, and 3. The Syntaxin-binding properties of the mutants were tested using an in vitroassay and by a co-immunoprecipitation approach employing Munc18b expressed in CHO-K1 cells. Three Munc18b variants, W28S, S42K, and E59K, were generated that are defective in binding to all three Syntaxins. A fourth mutant protein, S48D, shows abolishment of Syntaxin 3 interaction but binds Syntaxin 2 at normal and Syntaxin 1A at mildly reduced efficiency. Over-expression of Munc18b S48D inhibited transport of influenza hemagglutinin to the apical surface of Madin-Darby canine kidney II cells, which express Syntaxin 2 abundantly, but not of Caco-2 cells, in which Syntaxin 3 is the major apical target SNARE (soluble NSF (N-ethylmaleimide sensitive factor) attachment protein receptors). This suggests that, although Syntaxin 3 is the main target SNARE operating in exocytic transport to the apical plasma membrane in certain epithelial cell types, Syntaxin 2 may play an important role in this trafficking route in others.
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munc18 2 a functional partner of Syntaxin 3 controls apical membrane trafficking in epithelial cells
Journal of Biological Chemistry, 2000Co-Authors: Kirsi Riento, Maria Kauppi, Sirkka Keranen, Vesa M. OlkkonenAbstract:The Sec1-related proteins bind to Syntaxin family t-SNAREs with high affinity, thus controlling the interaction of Syntaxins with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with Syntaxin 3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions in conserved amino acid residues. The mutants displayed a spectrum of different Syntaxin binding efficiencies. The in vitro andin vivo binding patterns were highly similar, and the association of the Munc18-2 variants with Syntaxin 3 correlated well with their ability to displace SNAP-23 from Syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do not detectably bind Syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the Syntaxin interaction is not the sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind Syntaxin 3 behaved differently in the HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the Syntaxin 3 interaction.
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Syntaxin 3 and munc 18 2 in epithelial cells during kidney development
Kidney International, 1999Co-Authors: Sanna Lehtonen, Vesa M. Olkkonen, Kirsi Riento, Eero LehtonenAbstract:Syntaxin 3 and Munc-18-2 in epithelial cells during kidney development. Background Differentiation of epithelial cells involves the assembly of polarized membrane transport machineries necessary for the generation and maintenance of the apical and basolateral membrane domains characteristic of this cell type. We have analyzed the expression patterns of vesicle-docking proteins of the Syntaxin family in mouse kidney, focusing on Syntaxin 3 and its interaction partner, the Sec1-related Munc-18-2. Methods Expression patterns were studied by in situ hybridization and immunocytochemistry and the complex formation of Syntaxin 3 and Munc-18-2 by coimmunoprecipitation and Western blotting. Results We have previously shown by in situ hybridization that Munc-18-2 is present in the proximal tubules and collecting ducts of embryonic day 17 mouse kidney. We compared this with the expression patterns of Syntaxin 1A, 2, 3, 4, and 5, and found that Syntaxin 3 was enriched in the same epithelial structures in which Munc-18-2 was abundant. By immunocytochemistry, the two proteins colocalized at the apical plasma membrane of proximal tubule and collecting duct epithelial cells, and they were shown to form a physical complex in the kidney. The expression of both proteins was up-regulated during kidney development. The most prominent changes in expression levels coincided with the differentiation of proximal tubules, suggesting a role in the generation of the highly active reabsorption machinery characterizing this segment of the nephron. Conclusion The results show that Munc-18-2 and Syntaxin 3 form a complex in vivo and suggest that they participate in epithelial cell differentiation and targeted vesicle transport processes in the developing kidney.
