Jean-Claude Louis

Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [3H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met547 and Thr550) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [3H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders. Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [3H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met547 and Thr550) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [3H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders. The receptor for capsaicin (a small vanilloid molecule extracted from “hot” chili peppers), designated vanilloid receptor 1 (also known as VR1 and TRPV1 1The abbreviations used are: TRPV1, transient receptor potential vanilloid type 1; RTX, resiniferatoxin; AEA, arachidonyl ethanolamine; NADA, N-arachidonyldopamine; OLDA, oleoyldopamine; DRG, dorsal root ganglia; PPAHV, 12-phenylacetate 13-acetate 20-homovanillate; BCTC, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide; CHO, Chinese hamster ovary; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; r/o, rat-rabbit chimera; h/o, human-rabbit chimera; Iodo-RTX, iodo resiniferatoxin; TM, transmembrane domain. (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7310) Google Scholar)) has been cloned and shown to be a nonselective cation channel with high permeability to calcium. TRPV1 belongs to a superfamily of ion channels known as transient receptor potential channels (TRPs) several of which appear to be sensors of temperature (2Julius D. Basbaum A.I. Nature. 2001; 413: 203-210Crossref PubMed Scopus (1985) Google Scholar, 3Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 6: 387-396Crossref Scopus (982) Google Scholar). TRPV1 can be activated by exogenous agonists (capsaicin and RTX) and by physical stimuli such as heat (>42 °C) and protons (pH 5). Possible endogenous ligands released during tissue injury have also been suggested, including anandamide (arachidonylethanolamine or AEA) and products of lipoxygenases such as 12-hydroperoxyeicosatetraenoic acid, N-arachidonyldopamine (NADA), and N-oleoyldopamine (OLDA) (4Hwang S.W. Cho H. Kwak J. Lee S.Y. Kang C.J. Jung J. Cho S. Min K.H. Suh Y.G. Kim D. Oh U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6155-6160Crossref PubMed Scopus (976) Google Scholar, 5Olah Z. Karai L. Iadarola M.J. J. Biol. Chem. 2001; 276: 31163-31170Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 6Huang S.M. Bisogno T. Trevisani M. Al-Hayani A. De Petrocellis L. Fezza F. Tognetto M. Petros T.J. Krey J.F. Chu C.J. Miller J.D. Davies S.N. Geppetti P. Walker J.M. Di Marzo V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8400-8405Crossref PubMed Scopus (840) Google Scholar, 7Chu C.J. Huang S.M. De Petrocellis L. Bisogno T. Ewing S.A. Miller J.D. Zipkin R.E. Daddario N. Appendino G. Di Marzo V. Walker J.M. J. Biol. Chem. 2003; 278: 13633-13639Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Ji et al. (8Ji R.R. Samad T.A. Jin S.X. Schmoll R. Woolf C.J. Neuron. 2002; 36: 57-68Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar) reported that TRPV1 is detectable at increased levels after inflammatory injury in rodents and speculated that the increased level of TRPV1 protein combined with the confluence of stimuli present in inflammatory injury states leads to a reduced threshold of activation of nociceptors that express TRPV1, i.e. hyperalgesia. Indeed the converse is true that TRPV1-deficient mice display reduced thermal hypersensitivity following inflammatory tissue injury (9Caterina M.J. Leffler A. Malmberg J. M. Basbaum A.I. Julius D. 2000; PubMed Scopus Google Scholar). of channel in their but have been of binding of the TRPV1 [3H]RTX to dorsal root TRPV1 cloned A. PubMed Scopus Google Scholar). rabbits to be to the of capsaicin T. V. C. T. V. PubMed Scopus Google Scholar) and to have [3H]RTX binding A. PubMed Scopus Google Scholar). have the for to identify key regions in TRPV1 binding and activation by RTX and capsaicin by TRPV1 from and insensitive (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7310) Google human P. M.J. S. P. J. 2000; Full Text Full Text PDF PubMed Scopus Google rabbit S. R. S. R. J. J. Neurosci. 2000; Scholar, L. R. S. J. J. J. J. 2003; Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google and J. C. S. S. J. P. H. P. 2002; PubMed Scopus Google and human TRPV1 have been that capsaicin and RTX agonists of TRPV1 (capsaicin and RTX expressed in P. M.J. S. P. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, P. A. M. J. C. M. J. N. M. S. J. 2001; PubMed Scopus Google Scholar, S.W. Cho H. Cho S.Y. M.J. Lee S.Y. Oh U. Neurosci. 2001; PubMed Scopus Google Scholar). Interestingly, these have differences in such as the report that capsazepine human but rat TRPV1 response to P. A. M. J. C. M. J. N. M. S. J. 2001; PubMed Scopus Google Scholar). of capsaicin J. S.W. Kwak J. Lee Kang C.J. Kim Kim D. Oh U. J. Neurosci. PubMed Google Scholar) and of Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar, J.M. S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar) have that contribute to capsaicin and have that capsaicin to from the and protons on to TRPV1. We have reported the of rabbit TRPV1 and that it is but activated by heat °C) and protons (pH in expressed S. R. S. R. J. J. Neurosci. 2000; Scholar). and Julius Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) have shown that expressed TRPV1 is insensitive to activation by capsaicin but sensitive to heat (>42 °C) and (pH Furthermore, and Julius Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that the TM3/4 region of TRPV1 to be for capsaicin by have residues on the and of TRPV1 that also appear to capsaicin as well as [3H]RTX binding J. Lee S.W. Cho H. J. Kang Kim S. Oh U. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, V. J. A. R. L. J. Neurosci. 2003; PubMed Google Scholar). We in the present in TRPV1 for vanilloid by rabbit TRPV1 and of the TM3/4 We assays and the of oTRPV1 and to the of TRPV1. of [3H]RTX used to residues for the high binding of ligand to TRPV1. Furthermore, we the functional of oTRPV1 and to capsaicin antagonists and show that of capsaicin also competitive antagonist at TRPV1. we present a model of capsaicin and RTX bound to the TM3/4 region of rat TRPV1. in from extracted from dorsal root from The at high °C) with a the with and the designated for The of has been to the TRPV1 by and from and in and cloned oTRPV1 the following the by as by J. PubMed Scopus Google Scholar). of oTRPV1 the of with or to or at with the or to with and to of tissue and in with bovine and with a TRPV1 receptor by of and of in a the of for for for or or and used for [3H]RTX binding or with used to or by with TRPV1 in with bovine and The used for of in used as a of and for of TRPV1 in a and used in of our to activation of TRPV1 is as a of of the assays a at The assays as agonists with or TRPV1 in of and with BSA, and 1 at at temperature for in the of to antagonist with or TRPV1 or in with BSA, and 1 at at temperature for to of in and for to antagonist with or TRPV1 or at temperature for to of in and for to and with after functional in the a in response as for in the [3H]RTX with [3H]RTX as with A. PubMed Scopus Google Scholar). on in and of of binding of [3H]RTX and of The at a of of binding and binding in the of of RTX The at for 1 by the on for of the binding and for to binding. The bound and ligands by in a The of the the and the bound by the TRPV1 channels at the A. PubMed Scopus Google Scholar). The and with and The from to 4 and to by a a the a the and during the of at by a and at in at temperature °C) by the potential at model used to to the and to the by and and the of RTX and capsaicin and of TRPV1 transient in of by for TRPV1 protein that TRPV1 in report to be for of the of functional channels expressed on the and as such of the data as of assays TRPV1 that have the permeability to oTRPV1 also by in oTRPV1 to oTRPV1 is less sensitive to than oTRPV1 cloned from a of a rabbit with a protein high homology to and and and and of rabbit dorsal root with a from the oTRPV1 of in the with to the small and with that in that of a on oTRPV1 activated by heat °C) or to it activated by capsaicin at activation for Furthermore, show [3H]RTX binding with a of and homology between TRPV1 from is shown at the and is shown in the in a also by of TRPV1 with with that increased with at for and oTRPV1 a of in which that the in and by TRPV1. also in response to 1 and capsaicin in 1 capsaicin to in a small that oTRPV1 expressed in and that oTRPV1 less sensitive to activation by capsaicin than the small with capsaicin suggested a in The of to oTRPV1 also in after activation of the oTRPV1 data that activation of oTRPV1 is sensitive to to to from the activation of oTRPV1 by and of the and heat by and the of oTRPV1 in rabbit dorsal root that oTRPV1 is the rabbit of TRPV1. The limited of oTRPV1 to capsaicin and RTX and lack of detectable [3H]RTX binding with data on the of oTRPV1 A. PubMed Scopus Google Scholar, T. V. C. T. V. PubMed Scopus Google Scholar). for Vanilloid in rat-rabbit of TRPV1 by of transmembrane 3 4 from to and Julius Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that the TM3/4 region of TRPV1 to be for capsaicin of by that the to for and to of the to capsaicin also by by and 1 capsaicin in the and and we also a human-rabbit the from to to functional that to capsaicin that the TM3/4 region is for vanilloid of the region that between rat and rabbit TRPV1, and between human and rabbit TRPV1 which residues region for of functional to in we the residues that in rabbit from rat and human TRPV1 and the at in rabbit to the in rat and human TRPV1 to confer of for activation by capsaicin in that the of capsaicin at the oTRPV1 channel it for for the and for to the we have also to in or show to capsaicin or known TRPV1 agonists 12-phenylacetate 13-acetate NADA, and to at type oTRPV1 but as potent agonists at residues and or in in response of oTRPV1 to of capsaicin in by or capsaicin in and that vanilloid and of activation of TRPV1 and agonists in or for and expressed in The of the to RTX and to capsaicin is by that be the of response to RTX is to that for our of a to the of RTX may be to capsaicin, it the of for the TRPV1 in a the at we several and in capsaicin a of at of oTRPV1 of the at with the small in of capsaicin and with the in a small in oTRPV1 capsaicin capsaicin of and oTRPV1 their to activation to type oTRPV1 of with its at in a of TRPV1 response to or heat levels of to for Vanilloid in and that the in rat and human TRPV1 to vanilloid of TRPV1, we a of by the with the oTRPV1 in that the of capsaicin at the is to from at type channel, a in of capsaicin at the is to from at type channel, a in also of capsaicin in and 1 capsaicin to in (pH in that capsaicin of vanilloid with in oTRPV1 and with a in rat and human that is of the for TRPV1 activation by Based on their and Julius Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) reported that is for vanilloid is in TRPV1 from reported to We have that the is for vanilloid and vanilloid as shown by to 1 or capsaicin to to and we have capsaicin of oTRPV1 of and of i.e. with the in capsaicin of in is the in of capsaicin by is than the in as by the of the capsaicin the type oTRPV1 that is for vanilloid of TRPV1. Interestingly, of rat to the rabbit in the capsaicin to the it appear to RTX in the for and and [3H]RTX binding reduced in the we report a in the for the functional of TRPV1 to capsaicin and which is insensitive of with to capsaicin and RTX of in rat TRPV1 with present in rabbit TRPV1 in of functional response to the capsaicin, but to the high and binding of or with the a functional response to and vanilloid activation and data the to show [3H]RTX binding. the oTRPV1 the rat region binding with a of to that residues the region to [3H]RTX binding in between RTX in functional and binding assays has been reported A. Rev. Google Scholar, G. Lee J. V. PubMed Google Scholar). in A. Z. PubMed Scopus Google is that of the TRPV1 is and a small is to the The of TRPV1 at the the the binding is by the TRPV1, and these of TRPV1 display differences in the receptor for RTX in these assays and the of residues in TRPV1 for to the residues that between rat and we a of oTRPV1 and to the residues in rat TRPV1, which has been shown to display the RTX binding in TRPV1 to RTX as by of oTRPV1 residues at and to residues in and the oTRPV1 response to capsaicin or RTX Interestingly, the in oTRPV1 in a of to RTX with in capsaicin in assays and at oTRPV1 and and for RTX and and for capsaicin, that is of the residues to RTX to RTX in and expressed to show [3H]RTX binding We that to RTX but residues such as for for [3H]RTX binding the to RTX in a type and to capsaicin and RTX in the functional and at and and for RTX and and for capsaicin, the reduced [3H]RTX binding with type a between functional assays and binding and that to RTX in the binding and for [3H]RTX in in to RTX but to capsaicin in the functional a in of oTRPV1 to we to [3H]RTX binding in either of these and we that oTRPV1 and to [3H]RTX binding. We the oTRPV1 and its vanilloid in functional and [3H]RTX binding [3H]RTX binding with a of with a in functional to capsaicin and a for RTX, with of and 1 and that and as present in for [3H]RTX binding in of the oTRPV1 of and to protons or heat with oTRPV1 and with activated by and oTRPV1 activation at and from rat TRPV1. the residues in oTRPV1 to the residues in and in a in to by oTRPV1 from that of the type channel that oTRPV1 and the expressed and to stimuli their to of to have by in response to a of TRPV1 including the RTX, capsaicin, and PPAHV as well as the proposed endogenous and binding of [3H]RTX RTX oTRPV1 with of in the with 3 of NADA, OLDA, AEA, and PPAHV at oTRPV1 to (pH The of for RTX capsaicin PPAHV of capsaicin and RTX by and with their of to TRPV1 agonists capsaicin, NADA, The of for at sensitive oTRPV1 RTX capsaicin PPAHV (a at rat but human TRPV1 P. A. M. J. C. M. J. N. M. S. J. 2001; PubMed Scopus Google Scholar)) a of in oTRPV1 for to be is to and activation by PPAHV is is to and activation is that PPAHV and that is for RTX and which also the differences between and Vanilloid the of oTRPV1 and its we have at the of a of antagonists to of activation of these channel and capsaicin activation of and with of oTRPV1 is sensitive to capsaicin and and we to reported TRPV1 antagonists activation of type as well as and capsaicin activation of its We antagonists that reported to capsaicin and activation of human and rat TRPV1, i.e. and A. Z. PubMed Scopus Google Scholar, G. M. L. L. J. S. J. 2003; PubMed Scopus Google Scholar, J.D. L. Walker J. 2003; PubMed Scopus Google Scholar) as well as reported to but rat TRPV1 to P. A. M. J. C. M. J. N. M. S. J. 2001; PubMed Scopus Google of capsaicin and activation of TRPV1 antagonists in or activated by capsaicin or for antagonist and is expressed in or in at in at capsaicin in a in and potent antagonists of and activated by capsaicin for at at and at at 4 1 as well as by protons (pH for and at and at and of the antagonists to than 100-fold the activation of that oTRPV1 the key for binding of competitive antagonists such as and and We that of capsaicin and activation of also capsaicin and activation of and and by and in response to capsaicin or activation. and capsaicin activation of with of and and of with of and activation of with of and with of and activation of the with of and with of and for and of rabbit TRPV1 activated with capsaicin or that is for vanilloid but also for the of competitive antagonists to of TRPV1 channel activation. capsazepine of capsaicin at and oTRPV1 its of (pH activation pharmacological of to be with to capsazepine at activation. for capsazepine at activated and and but capsazepine to activated and and have at and have Based on these we propose that is for capsazepine of activation of TRPV1, it is that residues to human and rabbit TRPV1 region and also contribute to capsazepine of activated TRPV1. has been reported that membranes from rabbit dorsal root lack [3H]RTX binding in to to the for and of TRPV1, we cloned and a from a rabbit The reported to be of human TRPV1. oTRPV1 is expressed in rabbit dorsal root and to be to the small to to on rat and human TRPV1. Furthermore, oTRPV1 expressed in or can be activated by the physical stimuli and to rat and human TRPV1. as from the [3H]RTX binding oTRPV1 is insensitive to vanilloid agonists capsaicin, and as with rat and human TRPV1. to the of and Julius Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) in of the to region from rat or human TRPV1 vanilloid to oTRPV1 to the type rat and human TRPV1 as by assays and and Julius that a for vanilloid with of and is a it the differences we have in vanilloid in rabbit TRPV1 and of the in the residues the pharmacological of the rabbit TRPV1 channel by residues from the rat TRPV1 channel, and the channels have in the of which confer vanilloid and high [3H]RTX binding in and human TRPV1 we that oTRPV1 can functional vanilloid with a and we show that with oTRPV1 also high [3H]RTX binding. of the rat and human residues at and show of vanilloid RTX binding. Based on and on TRPV1 Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google and the present vanilloid and RTX binding with the TM3/4 region from rat TRPV1 to and rabbit as well as to such as and that the is by the TM3/4 is by the of and Julius Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google in which that a [3H]RTX binding to the in the TM3/4 and the present the of [3H]RTX binding by oTRPV1 and these the that and may be present in the binding and for vanilloid Based on these be proposed capsaicin or RTX with and and Julius Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that the of with the vanilloid of capsaicin their model for the capsaicin that with the TRPV1 from J. S.W. Kwak J. Lee Kang C.J. Kim Kim D. Oh U. J. Neurosci. PubMed Google model be proposed by vanilloid with and the of capsaicin or RTX with in the present in and for the differences in between ligands to the differences in the of these i.e. with such as may well with and agonists of TRPV1. the with such as and may with and for their is for capsaicin, RTX, and it is to that with of the by with the from of the vanilloid of oTRPV1 the of by and by and that and have the and in but lack the functional which is for and antagonist capsaicin with of and which display R. S. A. J. J. Chem. 36: PubMed Scopus Google Scholar) and antagonist belongs to the between and and residues and to Based on the of the channel and the model of the TM3/4 region Lee A. J. V. M. R. Nature. 2003; PubMed Scopus Google it is that TM3/4 regions of the ion channels such as TRPV1 may a and the binding may be as as the and with a to the vanilloid as may from model is on that the of ion and the by the TM3/4 region of TRPV1, which be for our in have to be but in The model and the model by and Julius Julius D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) account for and of TRPV1 V. J. A. R. L. J. Neurosci. 2003; PubMed Google Scholar) or at and J. Lee S.W. Cho H. J. Kang Kim S. Oh U. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) in of to capsaicin and a of binding to that residues in TM3/4 regions for ligand recognition and that in the and a and and information for and of ligand in the binding is that or with the vanilloid as be shown either by of these residues with the vanilloid or by a of capsaicin or RTX bound to TRPV1, model on be proposed to our and the of TRPV1 in a that vanilloid binding in the TM3/4 region that may model the from of the vanilloid R. S. A. J. J. Chem. 36: PubMed Scopus Google we the rabbit TRPV1, of to the rat in a to RTX a detectable in capsaicin RTX is a vanilloid of to capsaicin, is to be by the known as a The of the in oTRPV1 that to a that is for with Interestingly, the of the rabbit TRPV1 to the response to capsaicin and has from that the of binding to TRPV1, as by of [3H]RTX and of G. Lee J. V. PubMed Google Scholar). TRPV1 is expressed at membranes in the with a small at the it can for the in is that the TRPV1 channels present from at the its by Nature. 2000; PubMed Scopus Google Scholar, G. H. Neuron. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Tominaga T. H. Tominaga M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google with Julius D. 2003; PubMed Scopus Google or with M. Tominaga T. N. H. Tominaga M. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). for the of a vanilloid antagonist that in response to capsaicin or RTX with high of but which [3H]RTX binding less than at A. Z. PubMed Scopus Google Scholar). antagonist to the of from by The such pharmacological between the assays for and [3H]RTX binding. the present the is by in the receptor than in the we in the [3H]RTX binding that of rat TRPV1 from either to or to a in binding but a in for of to for [3H]RTX binding is with the that human TRPV1, which has for [3H]RTX binding with G. Lee J. V. PubMed Google Scholar). We also the of several of the and TRPV1 to the known We and G. M. L. L. J. S. J. 2003; PubMed Scopus Google Scholar, P. C. S. C. 2001; PubMed Scopus Google Scholar) have shown that and potent antagonists of capsaicin and activation of rat and human TRPV1. and antagonists of oTRPV1 to protons (pH 5). and in potent antagonists of oTRPV1 of and we that is also a for competitive antagonist binding. We that TRPV1 the of capsazepine with the binding model of capsaicin that the of the have as the of the the of antagonists to of TRPV1 activation and of of the key and Thr550) as a key on TRPV1 and may in antagonists with of the of TRPV1 in inflammatory and sensory (8Ji R.R. Samad T.A. Jin S.X. Schmoll R. Woolf C.J. Neuron. 2002; 36: 57-68Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, M.J. Leffler A. Malmberg J. M. Basbaum A.I. Julius D. 2000; PubMed Scopus Google antagonists of may in the treatment of human such as and We and for for and for NADA, and

The novel transmembrane aspartic protease BACE (for Beta-site APP Cleaving Enzyme) is the beta-secretase that cleaves amyloid precursor protein to initiate beta-amyloid formation. As such, BACE is a prime therapeutic target for the treatment of Alzheimer's disease. BACE, like other aspartic proteases, has a propeptide domain that is removed to form the mature enzyme. BACE propeptide cleavage occurs at the sequence RLPR downward arrowE, a potential furin recognition motif. Here, we explore the role of furin in BACE propeptide domain processing. BACE propeptide cleavage in cells does not appear to be autocatalytic, since an inactive D93A mutant of BACE is still cleaved appropriately. BACE and furin co-localize within the Golgi apparatus, and propeptide cleavage is inhibited by brefeldin A and monensin, drugs that disrupt trafficking through the Golgi. Treatment of cells with the calcium ionophore, leading to inhibition of calcium-dependent proteases including furin, or transfection with the alpha(1)-antitrypsin variant alpha(1)-PDX, a potent furin inhibitor, dramatically reduces cleavage of the BACE propeptide. Moreover, the BACE propeptide is not processed in the furin-deficient LoVo cell line; however, processing is restored upon furin transfection. Finally, in vitro digestion of recombinant soluble BACE with recombinant furin results in complete cleavage only at the established E46 site. Taken together, our results strongly suggest that furin, or a furin-like proprotein convertase, is responsible for cleaving the BACE propeptide domain to form the mature enzyme.