Thermal Stabilization of the Catalytic Domain of Botulinum Neurotoxin E by Phosphorylation of a Single Tyrosine Residue

Blanes-Mira, Clara; Ibáñez, Cristina Villalba; Fernández‐Ballester, Gregorio; Planells-Cases, Rosa; Pérez‐Payá, Enrique; Ferrer‐Montiel, Antonio

doi:10.1021/bi001919y

Cited by 31 publications

(62 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…BoNT A fully blocked TPA-induced TRPV1 surface expression. Western immunoblots were digitized and quantified as described (23). Values are mean Ϯ S.E., n ϭ 3 (*, p Ͻ 0.05; Student's t test).…”

Section: Discussionmentioning

confidence: 99%

“…After three washes with binding buffer, Ni 2ϩ -bound complexes were eluted from the resin and denatured with SDS-PAGE sample buffer at 90°C for 10 min. Proteins complexes were resolved by SDS/PAGE on 12% gels, visualized by autofluorography, and quantified as described (23).…”

Section: Methodsmentioning

confidence: 99%

“…Surface TRPV1 was detected by Western blotting with the anti-TRPV1 serum. Immunoblots were digitized and quantified as described (23).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Regulated Exocytosis Contributes to Protein Kinase C Potentiation of Vanilloid Receptor Activity

Morenilla‐Palao

Planells-Cases

García-Sanz

et al. 2004

Journal of Biological Chemistry

Self Cite

388

302

View full text Add to dashboard Cite

The vanilloid receptor-1 (TRPV1) plays a key role in the perception of peripheral thermal and inflammatory pain. TRPV1 expression and channel activity are notably up-regulated by proalgesic agents. The transduction pathways involved in TRPV1 sensitization are still elusive. We have used a yeast two-hybrid screen to identify proteins that associate with the N terminus of TRPV1. We report that two vesicular proteins, Snapin and synaptotagmin IX (Syt IX), strongly interact in vitro and in vivo with the TRPV1 N-terminal domain. In primary dorsal root ganglion neurons, TRPV1 co-distributes in vesicles with Syt IX and the vesicular protein synaptobrevin. Neither Snapin nor Syt IX affected channel function, but they notably inhibited protein kinase C (PKC)-induced potentiation of TRPV1 channel activity with a potency that rivaled the blockade evoked by botulinum neurotoxin A, a potent blocker of neuronal exocytosis. Noteworthily, we found that PKC activation induced a rapid delivery of functional TRPV1 channels to the plasma membrane. Botulinum neurotoxin A blocked the TRPV1 membrane translocation induced by PKC that was activated with a phorbol ester or the metabotropic glutamate receptor mGluR5. Therefore, our results indicate that PKC signaling promotes at least in part the SNARE-dependent exocytosis of TRPV1 to the cell surface. Taken together, these findings imply that activitydependent delivery of channels to the neuronal surface may contribute to the buildup and maintenance of thermal inflammatory hyperalgesia in peripheral nociceptor terminals.TRPV1 1 is a capsaicin-, proton-and heat-sensitive, cationselective ion channel expressed in nociceptors that participates in the transduction of noxious chemical and thermal stimuli by sensory nerve endings in peripheral tissues (1-3). Heterologous expression of TRPV1 cDNA results in ionic currents that recapitulate most of the functional properties displayed by native capsaicin-and heat-activated currents in sensory neurons (1, 2). For instance, TRPV1 exhibits a time-and Ca 2ϩ -dependent desensitization, a long lasting refractory state during which the receptor does not respond to vanilloids or other stimuli (2). In addition, the channel activity of TRPV1 is remarkably upregulated by inflammatory mediators through the activation of phospholipase C and protein kinases A and C (PKA and PKC) signaling pathways (4 -13). Recent evidence shows that an increase in TRPV1 expression in peripheral nociceptors is critical for the maintenance of inflammatory hyperalgesia (14, 15). The involvement of TRPV1 in heat hypersensitivity is further underscored by the reduced sensitivity of mice lacking TRPV1 (16, 17) and by mice treated with receptor-specific antagonists (18).TRPV1 belongs to the family of transient receptor potential channels, which structurally resembles the family of voltagegated potassium or cyclic nucleotide-gated channels (19). Accordingly, these channels are presumed to be tetrameric assemblies of identical subunits (Fig. 1A), although heteromeric assemblies have be...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Regulated Exocytosis Contributes to Protein Kinase C Potentiation of Vanilloid Receptor Activity

Morenilla‐Palao

Planells-Cases

García-Sanz

et al. 2004

Journal of Biological Chemistry

Self Cite

388

302

View full text Add to dashboard Cite

show abstract

“…The Ca^+ might allow the light chain to separate or change conformation as it enters the cytosol. 15 …”

Section: Role Of Metals In Botulinum Neurotoxinsmentioning

confidence: 99%

“…Though expression and purification of full-length BoNT/E-LC have been reported [14,15], the low quantity of protein that was recovered «1 mg/1) would require considerable scaleup to obtain sufficient material even to begin crystallization experiments. In this paper, we report the cloning of the full length BoNT/E-LC with a C-terminal 6x His-tag, in a pET-9c expression vector containing a bacteriophage T7 promoter.…”

Section: Bi035844kmentioning

confidence: 99%

Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexes with Inhibitors Leading to Drug Design

Swaminathan¹

2004

View full text Add to dashboard Cite

Public reporting burelen for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed and conpleting and reviewing this colleclion of infomiation. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this buiijen to Washington Headquarters Services.

show abstract

Aromatic side‐chain interactions in proteins: Near‐ and far‐sequence Tyr‐X pairs

Meurisse¹,

Brasseur²,

Thomas³

2003

Proteins

View full text Add to dashboard Cite

In the present study, an extensive analysis of the aromatic Tyr-X interactions is performed on a data set of 593 PDB structures, X being Phe, His, Tyr, and Trp. The nonredundant Tyr-X pairs (2645) were retained and separated by both the residue distance in the sequence and the secondary structures they bridge. Similar to the Phe-X and His-X pairs, the far-sequence Tyr-X pairs (X partner > five apart in the sequence: 74%) show comparable secondary structures and conformers for either type of X partner, in contrast with the near-sequence Tyr-X pairs (26%). As the Phe-X pairs, the near-sequence Tyr-X pairs stabilize secondary structures, mainly the alpha- helices (positions 1, 3, and 4) and the beta-strands (position 2). Like the Phe-X and His-X pairs, most far-sequence Tyr-X pairs (34%) bridge beta-strands and only 11% bridge helices. As for the Phe-X and the His-X pairs, the X partners of the far-sequence Tyr-X pairs are frequently "above" the tyrosine ring with tilted and normal rings, whereas the X partner of the near-sequence Tyr-X pairs gradually moves from the "aside" to the "above" location, together with a progressive decrease of normal and increase of parallel rings, respectively. Unlike the His-X pairs, the interactions of the hetroatom in Tyr-X pairs are only favored with a sequence position +4 and over, owing to the spatial accessibility of the heteroatom.

show abstract

Thermal Stabilization of the Catalytic Domain of Botulinum Neurotoxin E by Phosphorylation of a Single Tyrosine Residue

Cited by 31 publications

References 30 publications

Regulated Exocytosis Contributes to Protein Kinase C Potentiation of Vanilloid Receptor Activity

Regulated Exocytosis Contributes to Protein Kinase C Potentiation of Vanilloid Receptor Activity

Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexes with Inhibitors Leading to Drug Design

Aromatic side‐chain interactions in proteins: Near‐ and far‐sequence Tyr‐X pairs

Contact Info

Product

Resources

About