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1.
Front Med (Lausanne) ; 9: 934852, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36186812

RESUMO

A decline in the prevalence of parasites such as hookworms appears to be correlated with the rise in non-communicable inflammatory conditions in people from high- and middle-income countries. This correlation has led to studies that have identified proteins produced by hookworms that can suppress inflammatory bowel disease (IBD) and asthma in animal models. Hookworms secrete a family of abundant netrin-domain containing proteins referred to as AIPs (Anti-Inflammatory Proteins), but there is no information on the structure-function relationships. Here we have applied a downsizing approach to the hookworm AIPs to derive peptides of 20 residues or less, some of which display anti-inflammatory effects when co-cultured with human peripheral blood mononuclear cells and oral therapeutic activity in a chemically induced mouse model of acute colitis. Our results indicate that a conserved helical region is responsible, at least in part, for the anti-inflammatory effects. This helical region has potential in the design of improved leads for treating IBD and possibly other inflammatory conditions.

2.
J Nat Prod ; 83(11): 3454-3463, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33166137

RESUMO

Marine organisms produce a diverse range of toxins and bioactive peptides to support predation, competition, and defense. The peptide repertoires of stony corals (order Scleractinia) remain relatively understudied despite the presence of tentacles used for predation and defense that are likely to contain a range of bioactive compounds. Here, we show that a tentacle extract from the mushroom coral, Heliofungia actiniformis, contains numerous peptides with a range of molecular weights analogous to venom profiles from species such as cone snails. Using NMR spectroscopy and mass spectrometry we characterized a 12-residue peptide (Hact-1) with a new sequence (GCHYTPFGLICF) and well-defined ß-hairpin structure stabilized by a single disulfide bond. The sequence is encoded within the genome of the coral and expressed in the polyp body tissue. The structure present is common among toxins and venom peptides, but Hact-1 does not show activity against select examples of Gram-positive and Gram-negative bacteria or a range of ion channels, common properties of such peptides. Instead, it appears to have a limited effect on human peripheral blood mononuclear cells, but the ecological function of the peptide remains unknown. The discovery of this peptide from H. actiniformis is likely to be the first of many from this and related species.


Assuntos
Antozoários/química , Antibacterianos/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Peptídeos/farmacologia
3.
Biomolecules ; 10(8)2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32781704

RESUMO

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a "mini-granulin" structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents.


Assuntos
Granulinas/química , Dobramento de Proteína , Proteínas de Peixe-Zebra/química , Animais , Linhagem Celular , Proliferação de Células , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Granulinas/farmacologia , Humanos , Domínios Proteicos , Peixe-Zebra , Proteínas de Peixe-Zebra/farmacologia
4.
Biomedicines ; 8(5)2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32443665

RESUMO

Conopeptides belonging to the A-superfamily from the venomous molluscs, Conus, are typically α-conotoxins. The α-conotoxins are of interest as therapeutic leads and pharmacological tools due to their selectivity and potency at nicotinic acetylcholine receptor (nAChR) subtypes. Structurally, the α-conotoxins have a consensus fold containing two conserved disulfide bonds that define the two-loop framework and brace a helical region. Here we report on a novel α-conotoxin Pl168, identified from the transcriptome of Conus planorbis, which has an unusual 4/8 loop framework. Unexpectedly, NMR determination of its three-dimensional structure reveals a new structural type of A-superfamily conotoxins with a different disulfide-stabilized fold, despite containing the conserved cysteine framework and disulfide connectivity of classical α-conotoxins. The peptide did not demonstrate activity on a range of nAChRs, or Ca2+ and Na+ channels suggesting that it might represent a new pharmacological class of conotoxins.

5.
Front Pharmacol ; 10: 250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30949052

RESUMO

Chlorotoxin was originally isolated from the venom of the Israeli scorpion Leiurus quinquestriatus, and has potential as a tumor imaging agent based on its selective binding to tumor cells. Several targets have been suggested for chlorotoxin including voltage-gated chloride channels, and it has been shown to have anti-angiogenic activity and inhibit cell migration. The structure of chlorotoxin is stabilized by four disulfide bonds and contains ß-sheet and helical structure. Interestingly, the reduced form has previously been shown to inhibit cell migration to the same extent as the wild type, but structural analysis indicates that the reduced form of the peptide does not maintain the native secondary structure and appears unstructured in solution. This lack of structure suggests that a short stretch of amino acids might be responsible for the bioactivity. To explore this hypothesis, we have synthesized fragments of chlorotoxin without disulfide bonds. As expected for such small peptides, NMR analysis indicated that the peptides were unstructured in solution. However, the peptide corresponding to the eight C-terminal residues inhibited cell migration, in contrast to the other fragments. Our results suggest that the C-terminal region plays a critical role in the bioactivity of chlorotoxin.

6.
Biomedicines ; 6(4)2018 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-30301200

RESUMO

Inflammatory bowel diseases are a set of complex and debilitating diseases, for which there is no satisfactory treatment. Peptides as small as three amino acids have been shown to have anti-inflammatory activity in mouse models of colitis, but they are likely to be unstable, limiting their development as drug leads. Here, we have grafted a tripeptide from the annexin A1 protein into linaclotide, a 14-amino-acid peptide with three disulfide bonds, which is currently in clinical use for patients with chronic constipation or irritable bowel syndrome. This engineered disulfide-rich peptide maintained the overall fold of the original synthetic guanylate cyclase C agonist peptide, and reduced inflammation in a mouse model of acute colitis. This is the first study to show that this disulfide-rich peptide can be used as a scaffold to confer a new bioactivity.

7.
J Med Chem ; 61(19): 8746-8753, 2018 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-30183294

RESUMO

Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. Previously we reported that a truncated analogue ( Ov-GRN12-35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/ trans isomerization. To further investigate whether the proline residues are involved in adopting the multiple confirmations, we have synthesized analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity, and folding of Ov-GRN12-35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12-35_3s and might facilitate the development of a novel wound healing agent.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fasciola hepatica/química , Granulinas/farmacologia , Proteínas de Helminto/farmacologia , Fragmentos de Peptídeos/farmacologia , Dermatopatias/prevenção & controle , Cicatrização/efeitos dos fármacos , Animais , Fasciola hepatica/metabolismo , Fasciolíase/parasitologia , Feminino , Granulinas/química , Proteínas de Helminto/química , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/química , Conformação Proteica
8.
Mar Drugs ; 16(6)2018 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880743

RESUMO

The Australian jellyfish Chironex fleckeri, belongs to a family of cubozoan jellyfish known for their potent venoms. CfTX-1 and -2 are two highly abundant toxins in the venom, but there is no structural data available for these proteins. Structural information on toxins is integral to the understanding of the mechanism of these toxins and the development of an effective treatment. Two regions of CfTX-1 have been predicted to have helical structures that are involved with the mechanism of action. Here we have synthesized peptides corresponding to these regions and analyzed their structures using NMR spectroscopy. The peptide corresponding to the predicted N-terminal amphiphilic helix appears unstructured in aqueous solution. This lack of structure concurs with structural disorder predicted for this region of the protein using the Protein DisOrder prediction System PrDOS. Conversely, a peptide corresponding to a predicted transmembrane region is very hydrophobic, insoluble in aqueous solution and predicted to be structured by PrDOS. In the presence of SDS-micelles both peptides have well-defined helical structures showing that a membrane mimicking environment stabilizes the structures of both peptides and supports the prediction of the transmembrane region in CfTX-1. This is the first study to experimentally analyze the structure of regions of a C. fleckeri protein.


Assuntos
Venenos de Cnidários/química , Cubomedusas/química , Animais , Austrália , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Peptídeos/química
9.
J Biol Chem ; 292(24): 10288-10294, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28473469

RESUMO

Inflammatory bowel diseases (IBDs) are a set of complex and debilitating diseases for which there is no satisfactory treatment. Recent studies have shown that small peptides show promise for reducing inflammation in models of IBD. However, these small peptides are likely to be unstable and rapidly cleared from the circulation, and therefore, if not modified for better stability, represent non-viable drug leads. We hypothesized that improving the stability of these peptides by grafting them into a stable cyclic peptide scaffold may enhance their therapeutic potential. Using this approach, we have designed a novel cyclic peptide that comprises a small bioactive peptide from the annexin A1 protein grafted into a sunflower trypsin inhibitor cyclic scaffold. We used native chemical ligation to synthesize the grafted cyclic peptide. This engineered cyclic peptide maintained the overall fold of the naturally occurring cyclic peptide, was more effective at reducing inflammation in a mouse model of acute colitis than the bioactive peptide alone, and showed enhanced stability in human serum. Our findings suggest that the use of cyclic peptides as structural backbones offers a promising approach for the treatment of IBD and potentially other chronic inflammatory conditions.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Modelos Animais de Doenças , Fármacos Gastrointestinais/uso terapêutico , Modelos Moleculares , Peptídeos Cíclicos/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/imunologia , Colo/patologia , Desenho de Fármacos , Estabilidade de Medicamentos , Fármacos Gastrointestinais/síntese química , Fármacos Gastrointestinais/química , Humanos , Masculino , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Estabilidade Proteica , Proteólise , Distribuição Aleatória , Soro/enzimologia , Organismos Livres de Patógenos Específicos
10.
J Med Chem ; 60(10): 4258-4266, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28425707

RESUMO

Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as Ov-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant Ov-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of Ov-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin ß-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of ß-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from Ov-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas de Helminto/química , Proteínas de Helminto/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Opisthorchis/química , Cicatrização/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Helminto/síntese química , Proteínas de Helminto/uso terapêutico , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/síntese química , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Progranulinas , Alinhamento de Sequência
11.
Biophys J ; 110(8): 1744-1752, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27119635

RESUMO

Disulfide bridges are commonly found covalent bonds that are usually believed to maintain structural stability of proteins. Here, we investigate the influence of disulfide bridges on protein dynamics through molecular dynamics simulations on the cysteine-rich trypsin inhibitor MCoTI-II with three disulfide bridges. Correlation analysis of the reduced cyclic peptide shows that two of the three disulfide distances (Cys(11)-Cys(23) and Cys(17)-Cys(29)) are anticorrelated within ∼1 µs of bridge formation or dissolution: when the peptide is in nativelike structures and one of the distances shortens to allow bond formation, the other tends to lengthen. Simulations over longer timescales, when the denatured state is less structured, do not show the anticorrelation. We propose that the native state contains structural elements that frustrate one another's folding, and that the two bridges are critical for snapping the frustrated native structure into place. In contrast, the Cys(4)-Cys(21) bridge is predicted to form together with either of the other two bridges. Indeed, experimental chromatography and nuclear magnetic resonance data show that an engineered peptide with the Cys(4)-Cys(21) bridge deleted can still fold into its near-native structure even in its noncyclic form, confirming the lesser role of the Cys(4)-Cys(21) bridge. The results highlight the importance of disulfide bridges in a small bioactive peptide to bring together frustrated structure in addition to maintaining protein structural stability.


Assuntos
Dissulfetos/química , Peptídeos/química , Sequência de Aminoácidos , Ciclização , Simulação de Dinâmica Molecular , Conformação Proteica , Desnaturação Proteica , Temperatura
12.
Chembiochem ; 14(5): 617-24, 2013 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-23426877

RESUMO

Gomesin is an 18-residue peptide originally isolated from the hemocytes of the Brazilian spider Acanthoscurria gomesiana. A broad spectrum of bioactivities have been attributed to gomesin, including in vivo and in vitro cytotoxicity against tumour cells, antimicrobial, antifungal, anti-Leishmania and antimalarial effects. Given the potential therapeutic applications of gomesin, it was of interest to determine if an engineered version with a cyclic backbone has improved stability and bioactivity. Cyclization has been shown to confer enhanced stability and activity to a range of bioactive peptides and, in the case of a cone snail venom peptide, confer oral activity in a pain model. The current study demonstrates that cyclization improves the in vitro stability of gomesin over a 24 hour time period and enhances cytotoxicity against a cancer cell line without being toxic to a noncancerous cell line. In addition, antimalarial activity is enhanced upon cyclization. These findings provide additional insight into the influences of backbone cyclization on the therapeutic potential of peptides.


Assuntos
Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Sequência de Aminoácidos , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/toxicidade , Antimaláricos/síntese química , Antimaláricos/química , Antimaláricos/toxicidade , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Ciclização , Células HeLa , Humanos , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Aranhas/metabolismo
13.
Eur Biophys J ; 39(1): 111-20, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19305991

RESUMO

The hERG K(+) channel undergoes rapid inactivation that is mediated by 'collapse' of the selectivity filter, thereby preventing ion conduction. Previous studies have suggested that the pore-helix of hERG may be up to seven residues longer than that predicted by homology with channels with known crystal structures. In the present work, we determined structural features of a peptide from the pore loop region of hERG (residues 600-642) in both sodium dodecyl sulfate (SDS) and dodecyl phosphocholine (DPC) micelles using NMR spectroscopy. A complete structure calculation was done for the peptide in DPC, and the localization of residues inside the micelles were analysed by using a water-soluble paramagnetic reagent with both DPC and SDS micelles. The pore-helix in the hERG peptide was only two-four residues longer at the N-terminus, compared with the pore helices seen in the crystal structures of other K(+) channels, rather than the seven residues suggested from previous NMR studies. The helix in the peptide spanned the same residues in both micellar environments despite a difference in the localization inside the respective micelles. To determine if the extension of the length of the helix was affected by the hydrophobic environment in the two types of micelles, we compared NMR and X-ray crystallography results from a homologous peptide from the voltage gated potassium channel, KcsA.


Assuntos
Canais de Potássio Éter-A-Go-Go/química , Sequência de Aminoácidos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Porosidade , Estrutura Secundária de Proteína , Dodecilsulfato de Sódio/química
14.
J Biol Chem ; 284(2): 1000-8, 2009 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-18996846

RESUMO

Ion flow in many voltage-gated K(+) channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K(+) channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe(551) to Trp(563), affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala(561), His(562), Ala(565), Trp(568), and Ile(571)), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Sequência de Aminoácidos , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína
15.
J Biol Chem ; 283(14): 8969-75, 2008 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-18158286

RESUMO

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.


Assuntos
Aminoácidos/química , Isomerases/química , Peptídeos/química , Ornitorrinco , Proteínas/química , Peçonhas/enzimologia , Aminoácidos/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intercelular , Isomerases/genética , Isomerases/metabolismo , Mutação , Peptídeos/genética , Peptídeos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Estereoisomerismo , Especificidade por Substrato/fisiologia , Peçonhas/genética
16.
Biochemistry ; 46(11): 3065-74, 2007 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-17305365

RESUMO

Insulin-like growth factor binding proteins (IGFBPs) modulate the activity and distribution of insulin-like growth factors (IGFs). IGFBP-6 differs from other IGFBPs in being a relatively specific inhibitor of IGF-II actions. Another distinctive feature of IGFBP-6 is its unique N-terminal disulfide linkages; the N-domains of IGFBPs 1-5 contain six disulfides and share a conserved GCGCC motif, but IGFBP-6 lacks the two adjacent cysteines in this motif, so its first three N-terminal disulfide linkages differ from those of the other IGFBPs. The contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties and their structure-function relationships have been characterized in part, but the structure and function of the distinctive N-terminal subdomain of IGFBP-6 are unknown. Here we report the solution structure of a polypeptide corresponding to residues 1-45 of the N-terminal subdomain of IGFBP-6 (NN-BP-6). The extended structure of the N-terminal subdomain of IGFBP-6 is very different from that of the short two-stranded beta-sheet of the N-terminal subdomain of IGFBP-4 and, by implication, the other IGFBPs. NN-BP-6 contains a potential cation-binding motif; lanthanide ion binding was observed, but no significant interaction was found with physiologically relevant metal ions like calcium or magnesium. However, this subdomain of IGFBP-6 has a higher affinity for IGF-II than IGF-I, suggesting that it may contribute to the marked IGF-II binding preference of IGFBP-6. The extended structure and flexibility of this subdomain of IGFBP-6 could play a role in enhancing the rate of ligand association and thereby be significant in IGF recognition.


Assuntos
Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Somatomedinas/metabolismo , Sequência de Aminoácidos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Elementos da Série dos Lantanídeos/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/síntese química , Estrutura Terciária de Proteína
17.
Biochemistry ; 45(27): 8331-40, 2006 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-16819832

RESUMO

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated pl14a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. pl14a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an alpha-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of pl14a revealed a novel signal sequence, indicating that pl14a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of pl14a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, pl14a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50 = 1.59 microM) and neuronal (IC50 = 8.7 microM for alpha3beta4) and neuromuscular (IC50 = 0.54 microM for alpha1beta1 epsilondelta) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of pl14a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.


Assuntos
Conotoxinas/farmacologia , Caramujo Conus/química , Canal de Potássio Kv1.6/antagonistas & inibidores , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Conotoxinas/química , Conotoxinas/isolamento & purificação , Caramujo Conus/genética , DNA Complementar/química , Camundongos , Dados de Sequência Molecular , Músculos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/isolamento & purificação , Filogenia , Conformação Proteica
18.
J Biol Chem ; 281(33): 23668-75, 2006 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-16766795

RESUMO

SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI-1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp14 was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro13 and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active conformation of the binding loop.


Assuntos
Peptídeos Cíclicos/síntese química , Proteínas de Plantas/síntese química , Prolina/química , Inibidor da Tripsina de Soja de Bowman-Birk/síntese química , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Aprotinina/química , Bovinos , Sequência Conservada , Helianthus/química , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/genética , Proteínas de Plantas/genética , Prolina/genética , Inibidores de Serina Proteinase/química , Relação Estrutura-Atividade , Inibidor da Tripsina de Soja de Bowman-Birk/genética
19.
FEBS Lett ; 580(6): 1587-91, 2006 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-16480722

RESUMO

The presence of d-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d-amino-acid-residue isomerase(s) responsible for the modification of the all-l-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is approximately 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l-to-d-amino-acid-residue isomerase in a mammal.


Assuntos
Isomerases de Aminoácido/química , Ornitorrinco/metabolismo , Peçonhas/enzimologia , Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/isolamento & purificação , Sequência de Aminoácidos , Animais , Metanol/farmacologia , Dados de Sequência Molecular , Peptídeos/farmacologia
20.
Biochem Biophys Res Commun ; 340(4): 1098-103, 2006 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-16403439

RESUMO

The caseins (alphas1, alphas2, beta, and kappa) are phosphoproteins present in bovine milk that have been studied for over a century and whose structures remain obscure. Here we describe the chemical synthesis and structure elucidation of the N-terminal segment (1-44) of bovine kappa-casein, the protein which maintains the micellar structure of the caseins. kappa-Casein (1-44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nuclear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited considerable helical structure. Further, NMR analysis showed the presence of a helical segment containing 26 residues which extends from Pro8 to Arg34. This is the first report which demonstrates extensive secondary structure within the casein class of proteins.


Assuntos
Caseínas/química , Caseínas/ultraestrutura , Modelos Moleculares , Sequência de Aminoácidos , Animais , Caseínas/síntese química , Bovinos , Simulação por Computador , Cristalografia , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína
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