Clovis Nakaie - Academia.edu (original) (raw)

Papers by Clovis Nakaie

Research paper thumbnail of Synthesis and purification strategies for a long and hydrophobic transmembrane helix from rat bradykinin receptor

Research paper thumbnail of Interaction of bradykinin and its TOAC-containing derivatives with model

Research paper thumbnail of Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors

Peptides, Apr 1, 2013

Bradykinin (BK) and des-Arg 9-bradykinin (DBK) of kallikrein-kinin system exert its effects media... more Bradykinin (BK) and des-Arg 9-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B 2 (B 2 R) and B 1 (B 1 R) receptors, respectively. It was already shown that the deletion of kinin B 1 R or of B 2 R induces upregulation of the remaining receptor subtype [10,12,16,28,36]. However studies on overexpression of B 1 R or B 2 R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined [17,19,33]. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B 1 R (TGR(Tie 2 B 1)) exclusively in the endothelium. In another study, mice overexpressing B 1 R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharideinduced endotoxic shock. Therefore the role of B 2 R was investigated in the thoracic aorta isolated from TGR(Tie 2 B 1) rats overexpressing the B 1 R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B 2 R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie 2 B 1) rats could be due to the enhanced expression of B 2 R associated with the overexpression of the B 1 R.

Research paper thumbnail of Conformational properties of angiotensin II and its active and inactive TOAC-labeled analogs in the presence of micelles. Electron paramagnetic resonance, fluorescence, and circular dichroism studies

Biopolymers, 2009

The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6tetramethy... more The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents-negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC 1-AII and inactive TOAC 3-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOACpromoted intramolecular fluorescence quenching was more pronounced for TOAC 3-AII because of the proximity between the nitroxide and Tyr 4. CD spectra showed that although both AII and TOAC 1-AII presented flexible conformations in water, TOAC 3-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for AII and TOAC 1-AII, different conformations were acquired by TOAC 3-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC 3-AII is unable to acquire conformations similar to those of native AII and partially active TOAC 1-AII is probably the explanation for its lack of biological activity.

Research paper thumbnail of The Angiotensin II AT<sub>1</sub>Receptor Structure-Activity Correlations in the Light of Rhodopsin Structure

Physiological Reviews, Apr 1, 2007

I. Introduction 566 A. The Renin-angiotensin system 566 B. Scope of this review 567 II. Angiotens... more I. Introduction 566 A. The Renin-angiotensin system 566 B. Scope of this review 567 II. Angiotensin II Structure-Activity Relationships 567 A. Receptor binding and activation 567 B. Antagonism by ANG II analogs 568 C. Desensitization and tachyphylaxis 568 III. AT 1 Receptor Structure-Activity Correlations 569 A. Receptor structure 569 B. Receptor activation 573 C. Mechanisms following receptor activation 578 D. Overview of the AT 1 receptor 580 IV. Final Remarks 583 A. Active rhodopsin (AGPCR) structures as a result of probabilistic analyses 583 B. The extracellular locus as a site for cross-talking mechanisms 584 C. The AT 2 receptor is a natural constitutively activated form of AGPCR 584 D. The cluster of polar residues: the sodium site 584 V. Summary 584

Research paper thumbnail of Spectroscopic Studies of the Interaction of Native and TOAC-Labeled Peptide Hormones with Model Membranes: Angiotensin II

Biophysical Journal, 2010

Water transport through very narrow channels occurs according to the single file mechanism. While... more Water transport through very narrow channels occurs according to the single file mechanism. While entering the channel, every water molecule loses most of its neighbouring water molecules. The energetic costs are thought to be

Research paper thumbnail of Tachyphylactic properties of angiotensin II analogs with bulky and hydrophobic substituents at the N-terminus

Journal of Peptide Research, Oct 8, 2003

Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimula... more Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimulations with angiotensin II (Ang II), has been shown to be dependent mainly on the N-terminal region of the ligand. To further study the structural requirements for the induction of tachyphylaxis we have synthesized Ang II analogs containing the bulky and very lipophilic substituents 9-fluorenylmethyloxycarbonyl (Fmoc) and 9-fluorenylmethyl ester (OFm) at the alpha-amino (Nalpha-Fmoc-Ang II) or the beta-carboxyl ([Asp(OFm)1]-Ang II) groups of the Asp1 residue, respectively. In binding assays with Chinese hamster ovary cells transfected with the AT1 Ang II receptor, Nalpha-Fmoc-Ang II bound with high affinity, whereas [Asp(OFm)1]-Ang II showed lower affinity. In biological assays, these two analogs were full agonists and showed 30 and 3%, respectively, of the Ang II potency in contracting the guinea-pig ileum smooth muscle. The two analogs induced tachyphylaxis, in spite of the lack of a free amino group in Nalpha-Fmoc-Ang II. Thus, analogs with Fmoc- or OFm-type groups coupled to the Asp1 residue, whether at the amino or carboxyl functions, induce tachyphylaxis through an unreported mechanism. Based in these findings and those available from the literature, an alternate molecular interaction mode between Ang II N-terminal portion and the AT1 receptor is proposed to explain the tachyphylactic phenomenon.

Research paper thumbnail of Structure analysis of the second transmembrane segment of the rat bradykinin receptor in solution and in micelles by CD and fluorescence spectroscopies

Kluwer Academic Publishers eBooks, Mar 24, 2006

In a recent paper [1], we investigated alternative strategies to minimize difficulties in synthes... more In a recent paper [1], we investigated alternative strategies to minimize difficulties in synthesizing and purifying a long and hydrophobic transmembrane peptide sequence (Ac-CTVAEIYLGNLAGADLILASGLPFWAITANNFDNH2 , denoted TM2-34) corresponding to the (64–97) region of the second transmembrane segment of the rat BKB2 receptor [2]. Improved yield of synthesis was obtained by changing the solvent system during the coupling step, accordingly to our conjugated resin solvation-polarity of the medium strategy [3]. We also performed a preliminary conformational analysis of this 34-mer peptide and its minor fragment (74–94), denoted TM2-24, by circular dichroism (CD) in aqueous solution containing different amounts of hexafluoroisopropanol (HFIP). In this solvent, a well-known α-helical structure inducer, we have found a α-helical content lower than expected for this type of transmembrane fragment. Hence, to better investigate structural properties of these two peptides, taken as model of transmembrane fragment, CD and fluorescence spectroscopic methods were applied in aqueous solution, varying the pH and the proportion of trifluoroethanol (TFE), and an other secondary structure inducing solvent. This structural investigation was also carried out in presence of dodecylsulp h a t e ( S D S ) a n d N -hexadecyl-N,N,dimethyl-3-ammonium-1 propylsulphate(HPS)-type micelles. Owing to the low solubility of TM2-34 analog, most of experiments in aqueous solution were performed with the TM2-24 transmembrane fragment.

Research paper thumbnail of Structural studies of the antimicrobial peptide Tritrpticin (TRP3) by NMR

Programas e Resumos, 2006

Research paper thumbnail of EPR and CD conformational studies of toac-labeled bradykinin analogues in SDS and HPS model membranes

Research paper thumbnail of The angiotensin II A'T IND. 1A' receptor - a fluorescence quenching study of the depth of insertion of the receptor's second intra-cellular loop (IL2) in model membranes

Research paper thumbnail of Orientation of GPCR fragments (loops and transmembrane helices) in lipid membranes

Journal of Peptide Science, 2002

Research paper thumbnail of Synthesis of aggregating peptide sequences in highly-substituted resins: an esr approach

Research paper thumbnail of Fluorescence and EPR studies between model membranes and indolicidin, a Trp-rich antimicrobial peptide, and its spin-labeled analogue TOAC0-IND

Research paper thumbnail of Conformational Study of Cytoplasmic Domains of the Hamster a1B-Adrenergic Receptor

Research paper thumbnail of Conformational properties of extracellular and cytosolic loops of a G protein-coupled receptor

Research paper thumbnail of 膜貫通ブラジキニン受容体セグメントの化学集合体モニタリング 樹脂溶媒和,ペプチド鎖運動性とカップリング速度の相関関係

European Journal of Organic Chemistry, 2002

Research paper thumbnail of Reações de clivagem ácida de aminoácidos e peptídeos ligados a polímeros: relevância para a metodologia de síntese de peptídeo

Biotecnologia Aplicada à Agro&Indústria - Vol. 4, 2017

Os primeiros peptídeos foram sintetizados na década de 1950 1,2 , e a metodologia empregada naque... more Os primeiros peptídeos foram sintetizados na década de 1950 1,2 , e a metodologia empregada naquela época foi denominada "clássica", ou, mais comumente, em solução. Nesse método, as etapas sintéticas são efetuadas em solução homogênea, e ele envolve, geralmente, etapas de purificação de cada intermediário obtido, o que significa laboriosos processos de extrações,

Research paper thumbnail of Produção de peptídeos: aprimoramento da síntese em polímeros através do uso da ressonância paramagnética eletrônica (RPE)

Biotecnologia Aplicada à Agro&Indústria - Vol. 4, 2017

No mundo contemporâneo, a síntese química de peptídeos tem obtido cada vez mais importância devid... more No mundo contemporâneo, a síntese química de peptídeos tem obtido cada vez mais importância devido à contínua descoberta de inúmeras atividades dessas macromoléculas tanto no organismo animal quanto vegetal. Embora existam outras metodologias que levem à produção desse tipo de macromolécula, e que mencionaremos adiante, a mais empregada por

Research paper thumbnail of The role of N-terminal and C-terminal Arg residues from BK on interaction with kinin B2 receptor

Biological Chemistry, 2016

Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilata... more Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilatation, increase in vascular permeability and release of inflammatory mediators. BK performs its actions by coupling to and activating the B

Research paper thumbnail of Synthesis and purification strategies for a long and hydrophobic transmembrane helix from rat bradykinin receptor

Research paper thumbnail of Interaction of bradykinin and its TOAC-containing derivatives with model

Research paper thumbnail of Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors

Peptides, Apr 1, 2013

Bradykinin (BK) and des-Arg 9-bradykinin (DBK) of kallikrein-kinin system exert its effects media... more Bradykinin (BK) and des-Arg 9-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B 2 (B 2 R) and B 1 (B 1 R) receptors, respectively. It was already shown that the deletion of kinin B 1 R or of B 2 R induces upregulation of the remaining receptor subtype [10,12,16,28,36]. However studies on overexpression of B 1 R or B 2 R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined [17,19,33]. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B 1 R (TGR(Tie 2 B 1)) exclusively in the endothelium. In another study, mice overexpressing B 1 R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharideinduced endotoxic shock. Therefore the role of B 2 R was investigated in the thoracic aorta isolated from TGR(Tie 2 B 1) rats overexpressing the B 1 R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B 2 R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie 2 B 1) rats could be due to the enhanced expression of B 2 R associated with the overexpression of the B 1 R.

Research paper thumbnail of Conformational properties of angiotensin II and its active and inactive TOAC-labeled analogs in the presence of micelles. Electron paramagnetic resonance, fluorescence, and circular dichroism studies

Biopolymers, 2009

The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6tetramethy... more The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents-negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC 1-AII and inactive TOAC 3-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOACpromoted intramolecular fluorescence quenching was more pronounced for TOAC 3-AII because of the proximity between the nitroxide and Tyr 4. CD spectra showed that although both AII and TOAC 1-AII presented flexible conformations in water, TOAC 3-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for AII and TOAC 1-AII, different conformations were acquired by TOAC 3-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC 3-AII is unable to acquire conformations similar to those of native AII and partially active TOAC 1-AII is probably the explanation for its lack of biological activity.

Research paper thumbnail of The Angiotensin II AT<sub>1</sub>Receptor Structure-Activity Correlations in the Light of Rhodopsin Structure

Physiological Reviews, Apr 1, 2007

I. Introduction 566 A. The Renin-angiotensin system 566 B. Scope of this review 567 II. Angiotens... more I. Introduction 566 A. The Renin-angiotensin system 566 B. Scope of this review 567 II. Angiotensin II Structure-Activity Relationships 567 A. Receptor binding and activation 567 B. Antagonism by ANG II analogs 568 C. Desensitization and tachyphylaxis 568 III. AT 1 Receptor Structure-Activity Correlations 569 A. Receptor structure 569 B. Receptor activation 573 C. Mechanisms following receptor activation 578 D. Overview of the AT 1 receptor 580 IV. Final Remarks 583 A. Active rhodopsin (AGPCR) structures as a result of probabilistic analyses 583 B. The extracellular locus as a site for cross-talking mechanisms 584 C. The AT 2 receptor is a natural constitutively activated form of AGPCR 584 D. The cluster of polar residues: the sodium site 584 V. Summary 584

Research paper thumbnail of Spectroscopic Studies of the Interaction of Native and TOAC-Labeled Peptide Hormones with Model Membranes: Angiotensin II

Biophysical Journal, 2010

Water transport through very narrow channels occurs according to the single file mechanism. While... more Water transport through very narrow channels occurs according to the single file mechanism. While entering the channel, every water molecule loses most of its neighbouring water molecules. The energetic costs are thought to be

Research paper thumbnail of Tachyphylactic properties of angiotensin II analogs with bulky and hydrophobic substituents at the N-terminus

Journal of Peptide Research, Oct 8, 2003

Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimula... more Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimulations with angiotensin II (Ang II), has been shown to be dependent mainly on the N-terminal region of the ligand. To further study the structural requirements for the induction of tachyphylaxis we have synthesized Ang II analogs containing the bulky and very lipophilic substituents 9-fluorenylmethyloxycarbonyl (Fmoc) and 9-fluorenylmethyl ester (OFm) at the alpha-amino (Nalpha-Fmoc-Ang II) or the beta-carboxyl ([Asp(OFm)1]-Ang II) groups of the Asp1 residue, respectively. In binding assays with Chinese hamster ovary cells transfected with the AT1 Ang II receptor, Nalpha-Fmoc-Ang II bound with high affinity, whereas [Asp(OFm)1]-Ang II showed lower affinity. In biological assays, these two analogs were full agonists and showed 30 and 3%, respectively, of the Ang II potency in contracting the guinea-pig ileum smooth muscle. The two analogs induced tachyphylaxis, in spite of the lack of a free amino group in Nalpha-Fmoc-Ang II. Thus, analogs with Fmoc- or OFm-type groups coupled to the Asp1 residue, whether at the amino or carboxyl functions, induce tachyphylaxis through an unreported mechanism. Based in these findings and those available from the literature, an alternate molecular interaction mode between Ang II N-terminal portion and the AT1 receptor is proposed to explain the tachyphylactic phenomenon.

Research paper thumbnail of Structure analysis of the second transmembrane segment of the rat bradykinin receptor in solution and in micelles by CD and fluorescence spectroscopies

Kluwer Academic Publishers eBooks, Mar 24, 2006

In a recent paper [1], we investigated alternative strategies to minimize difficulties in synthes... more In a recent paper [1], we investigated alternative strategies to minimize difficulties in synthesizing and purifying a long and hydrophobic transmembrane peptide sequence (Ac-CTVAEIYLGNLAGADLILASGLPFWAITANNFDNH2 , denoted TM2-34) corresponding to the (64–97) region of the second transmembrane segment of the rat BKB2 receptor [2]. Improved yield of synthesis was obtained by changing the solvent system during the coupling step, accordingly to our conjugated resin solvation-polarity of the medium strategy [3]. We also performed a preliminary conformational analysis of this 34-mer peptide and its minor fragment (74–94), denoted TM2-24, by circular dichroism (CD) in aqueous solution containing different amounts of hexafluoroisopropanol (HFIP). In this solvent, a well-known α-helical structure inducer, we have found a α-helical content lower than expected for this type of transmembrane fragment. Hence, to better investigate structural properties of these two peptides, taken as model of transmembrane fragment, CD and fluorescence spectroscopic methods were applied in aqueous solution, varying the pH and the proportion of trifluoroethanol (TFE), and an other secondary structure inducing solvent. This structural investigation was also carried out in presence of dodecylsulp h a t e ( S D S ) a n d N -hexadecyl-N,N,dimethyl-3-ammonium-1 propylsulphate(HPS)-type micelles. Owing to the low solubility of TM2-34 analog, most of experiments in aqueous solution were performed with the TM2-24 transmembrane fragment.

Research paper thumbnail of Structural studies of the antimicrobial peptide Tritrpticin (TRP3) by NMR

Programas e Resumos, 2006

Research paper thumbnail of EPR and CD conformational studies of toac-labeled bradykinin analogues in SDS and HPS model membranes

Research paper thumbnail of The angiotensin II A'T IND. 1A' receptor - a fluorescence quenching study of the depth of insertion of the receptor's second intra-cellular loop (IL2) in model membranes

Research paper thumbnail of Orientation of GPCR fragments (loops and transmembrane helices) in lipid membranes

Journal of Peptide Science, 2002

Research paper thumbnail of Synthesis of aggregating peptide sequences in highly-substituted resins: an esr approach

Research paper thumbnail of Fluorescence and EPR studies between model membranes and indolicidin, a Trp-rich antimicrobial peptide, and its spin-labeled analogue TOAC0-IND

Research paper thumbnail of Conformational Study of Cytoplasmic Domains of the Hamster a1B-Adrenergic Receptor

Research paper thumbnail of Conformational properties of extracellular and cytosolic loops of a G protein-coupled receptor

Research paper thumbnail of 膜貫通ブラジキニン受容体セグメントの化学集合体モニタリング 樹脂溶媒和,ペプチド鎖運動性とカップリング速度の相関関係

European Journal of Organic Chemistry, 2002

Research paper thumbnail of Reações de clivagem ácida de aminoácidos e peptídeos ligados a polímeros: relevância para a metodologia de síntese de peptídeo

Biotecnologia Aplicada à Agro&Indústria - Vol. 4, 2017

Os primeiros peptídeos foram sintetizados na década de 1950 1,2 , e a metodologia empregada naque... more Os primeiros peptídeos foram sintetizados na década de 1950 1,2 , e a metodologia empregada naquela época foi denominada "clássica", ou, mais comumente, em solução. Nesse método, as etapas sintéticas são efetuadas em solução homogênea, e ele envolve, geralmente, etapas de purificação de cada intermediário obtido, o que significa laboriosos processos de extrações,

Research paper thumbnail of Produção de peptídeos: aprimoramento da síntese em polímeros através do uso da ressonância paramagnética eletrônica (RPE)

Biotecnologia Aplicada à Agro&Indústria - Vol. 4, 2017

No mundo contemporâneo, a síntese química de peptídeos tem obtido cada vez mais importância devid... more No mundo contemporâneo, a síntese química de peptídeos tem obtido cada vez mais importância devido à contínua descoberta de inúmeras atividades dessas macromoléculas tanto no organismo animal quanto vegetal. Embora existam outras metodologias que levem à produção desse tipo de macromolécula, e que mencionaremos adiante, a mais empregada por

Research paper thumbnail of The role of N-terminal and C-terminal Arg residues from BK on interaction with kinin B2 receptor

Biological Chemistry, 2016

Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilata... more Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilatation, increase in vascular permeability and release of inflammatory mediators. BK performs its actions by coupling to and activating the B