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1.
Artigo em Inglês | MEDLINE | ID: mdl-33825275

RESUMO

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, triply palmitoylated at the C-terminus region and supposed to be inserted into the lipid bilayer forming a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into 5 peptide segments and each of them was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, the O-acyl isopeptide structures were incorporated. After the ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted to the native peptide bond. Finally, the obtained polypeptide was successfully inserted into the bicelle, thus showing the success of the synthesis.

2.
Chem Commun (Camb) ; 56(91): 14239-14242, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33118552

RESUMO

Ferredoxin (Fd) is an electron carrier protein containing a [2Fe-2S] cluster. In this paper, we synthesized Se-Fd, in which four Cys residues coordinated to the cluster are substituted to selenocysteine. After the one-pot segment coupling by the thioester method, followed by deprotection and cluster loading, the desired Se-Fd was successfully obtained.

3.
J Biol Chem ; 295(27): 8914-8927, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32376688

RESUMO

Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue-long ß-amyloid peptide (Aß40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aß42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel ß-sheet, whereas previous studies by other researchers have reported parallel ß-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.

4.
Nat Commun ; 11(1): 1222, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144273

RESUMO

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Ubiquitinação , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Humanos , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Xenopus laevis
6.
J Org Chem ; 85(3): 1458-1465, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31793784

RESUMO

A prompt preparation method of the Fmoc-aminoacyl-N-alkylcysteine dipeptide by an Ugi four-component condensation reaction is described. Through a reaction with a commercially available Fmoc-amino acid, an amine, an isocyanide, and a mercaptoacetaldehyde derivative, one step synthesis of dipeptides containing 20 kinds of natural amino acid residues was achieved, which avoided the problematic N-alkylation of S-tritylcysteine and its coupling reaction. The dipeptide was applied to the Fmoc-solid-phase peptide synthesis, and peptide thioesters were successfully obtained in high efficiency via N-alkylcysteine (NAC)-assisted thioesterification.

7.
Chem Commun (Camb) ; 56(6): 956-959, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31858094

RESUMO

On-resin intramolecular native chemical ligation (NCL) assisted by N-ethylcysteine using Fmoc/SPPS to obtain cyclic peptides is described. N-terminal cysteine-containing peptides were subjected to NCL conditions leading to cyclization-cleavage reactions and consecutive S → N shift, rendering cyclic peptides in good yields and purities. The compounds were evaluated against P. falciparum 3D7.


Assuntos
Peptídeos Cíclicos/síntese química , Compostos de Sulfidrila/química , Cisteína/análogos & derivados , Cisteína/química , Estrutura Molecular , Peptídeos Cíclicos/química , Resinas Sintéticas/química
8.
Genes Cells ; 25(1): 22-32, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31680384

RESUMO

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Histonas/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Replicação do DNA , Histonas/fisiologia , Humanos , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
J Pept Sci ; 25(9): e3200, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31309659

RESUMO

Posttranslational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitination is one such modification. We previously reported on the synthesis of ubiquitinated histone H3 with an isopeptide mimetic structure. In this report, we describe the preparation of ubiquitinated histone H3 peptides with a native isopeptide structure, which showed a slightly weaker effect on the enzymatic activity of DNA methyltransferase 1 than the previous ubiquitinated H3 peptide analogs. These findings show that a native structure is important for determining the mechanism of the function, although ubiquitinated H3 peptide analogs can mimic the role of the original ubiquitinated H3. We also report on the successful preparation of the ubiquitinated full length histone H3.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Histonas/química , Peptídeos/farmacologia , Humanos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Ubiquitinação
10.
Biochem Biophys Res Commun ; 507(1-4): 242-245, 2018 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-30424878

RESUMO

To validate the use of recombinant aequorin (reAequorin) as a light emission standard, the protein concentrations of highly purified reAequorin were determined by amino acid composition analysis, and the presence of active reAequorin was confirmed by the ratio of absorbance peak at 460 nm to that at 280 nm. The high correlation of the luminescence intensity with the protein concentration showed that reAequorin could be used for a light emission standard to study the luminescence properties of luciferases and to evaluate the detection sensitivity of luminometers. The specific activity of Gaussia luciferase with Imax was 7.5-fold higher than that of reAequorin and was calculated to be 3.8 × 1016 quanta/mg protein.


Assuntos
Equorina/metabolismo , Copépodes/enzimologia , Luz , Luciferases/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Luminescência , Padrões de Referência
11.
Chem Commun (Camb) ; 54(83): 11737-11740, 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30276373

RESUMO

Selenocysteine-containing cyclic 8-mer peptides, which were designed to mimic the plausible catalytic tetrad of glutathione peroxidase, were successfully synthesized in one pot via tandem N-to-S acyl migration of N-alkylcysteine (NAC)-containing selenopeptides and intramolecular selenocysteine-mediated native chemical ligation (Sec-NCL) of the generated thioesters.

12.
Biochemistry ; 57(37): 5415-5426, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30148614

RESUMO

Although ovalbumin (OVA), a main component of hen egg white and a non-inhibitory serpin superfamily protein, has been reported to form fibrillar aggregates, its relationship with amyloid fibrils associated with various degenerative diseases is unclear. We studied the heat-induced aggregation of intact OVA using an amyloid-specific thioflavin T assay with a fluorometer or direct imaging with a light-emitting diode lamp and several physicochemical approaches, and the results confirmed that intact OVA forms aggregates with a small part of amyloid cores and dominantly amorphous aggregates. We isolated the amyloidogenic core peptide by proteolysis with trypsin. The isolated 23-residue peptide, pOVA, with marked amyloidogenicity, corresponded to one (ß-strand 3A) of the key regions involved in serpin latency transition and domain-swap polymerization leading to serpinopathies. Although the strong amyloidogenicity of pOVA was suppressed in a mixture of tryptic digests, it was observed under acidic conditions in the presence of various salts, with which pOVA has a positive charge. Cytotoxicity measurements suggested that, although heat-treated OVA aggregates exhibited the strongest toxicity, it was attributed to a general property of amorphous aggregates rather than amyloid toxicity. Predictions indicated that the high amyloidogenicity of the ß-strand 3A region is common to various serpins. This suggests that the high amyloidogenicity of ß-strand 3A that is important for serpin latency transition and domain-swap polymerization is retained in OVA and constitutes ß-spine amyloid cores upon heat aggregation.


Assuntos
Amiloide/farmacologia , Neoplasias do Colo/patologia , Temperatura Alta , Ovalbumina/química , Agregados Proteicos , Serpinas/química , Amiloide/química , Animais , Galinhas , Neoplasias do Colo/tratamento farmacológico , Camundongos , Polimerização , Células Tumorais Cultivadas
13.
Chemistry ; 24(11): 2593-2597, 2018 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-29314327

RESUMO

Amino acids bearing 4-methylbenzyl (MBn) and 4-methoxybenzyl (MPM)-protected sialic acid were synthesized and used for the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of a glycopeptide. The α-sialyl linkage of the MBn-protected unit was partially cleaved under the final deprotection by trifluoroacetic acid (TFA). In addition, the removal of several MBn groups were incomplete. On the other hand, the MPM-protected unit gave the desired glycopeptide without decomposition of the α-sialyl linkage. Using this unit, peptide thioesters of the tandem repeat unit of MUC1 mucin were synthesized by the N-alkylcysteine (NAC) method and used for the one-pot ligation by the thioester method. As a result, the three tandem repeats of MUC1 carrying sialyl Tn antigens were successfully synthesized.


Assuntos
Glicopeptídeos/química , Fluorenos/química , Glicopeptídeos/síntese química , Ácido N-Acetilneuramínico/química , Técnicas de Síntese em Fase Sólida , Ácido Trifluoracético/química
14.
J Comput Aided Mol Des ; 31(12): 1039-1052, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29147837

RESUMO

Single amino acid potential (SAAP) would be a prominent factor to determine peptide conformations. To prove this hypothesis, we previously developed SAAP force field for molecular simulation of polypeptides. In this study, the force field was renovated to SAAP3D force field by applying more accurate three-dimensional main-chain parameters, instead of the original two-dimensional ones, for the amino acids having a long side-chain. To demonstrate effectiveness of the SAAP3D force field, replica-exchange Monte Carlo (REMC) simulation was performed for two benchmark short peptides, chignolin (H-GYDPETGTWG-OH) and C-peptide (CHO-AETAAAKFLRAHA-NH2). For chignolin, REMC/SAAP3D simulation correctly produced native ß-turn structures, whose minimal all-atom root-mean-square deviation value measured from the native NMR structure (except for H) was 1.2 Å, at 300 K in implicit water, along with misfolded ß-hairpin structures with unpacked aromatic side chains of Tyr2 and Trp9. Similar results were obtained for chignolin analog [G1Y,G10Y], which folded more tightly to the native ß-turn structure than chignolin did. For C-peptide, on the other hand, the α-helix content was larger than the ß content on average, suggesting a significant helix-forming propensity. When the imidazole side chain of His12 was protonated (i.e., [His12Hip]), the α content became larger. These observations as well as the representative structures obtained by clustering analysis were in reasonable agreement not only with the structures of C-peptide that were determined in this study by NMR in 30% CD3CD in H2O at 298 K but also with the experimental and theoretical behaviors having been reported for protonated C-peptide. Thus, accuracy of the SAAP force field was improved by applying three-dimensional main-chain parameters, supporting prominent importance of SAAP for peptide conformations.


Assuntos
Peptídeo C/química , Simulação por Computador , Modelos Moleculares , Oligopeptídeos/química , Método de Monte Carlo , Conformação Proteica
15.
Mol Cell ; 68(2): 350-360.e7, 2017 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-29053958

RESUMO

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.


Assuntos
DNA (Citosina-5-)-Metiltransferases/química , Metilação de DNA , Histonas/química , Ubiquitina/química , Animais , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína , Ubiquitina/genética , Ubiquitina/metabolismo , Xenopus laevis
16.
Angew Chem Int Ed Engl ; 56(49): 15708-15711, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29048715

RESUMO

The synthesis of a peptide selenoester was efficiently carried out by the 9-fluorenylmethoxycarbonyl (Fmoc) method using N-alkylcysteine, at the C-terminus of the peptide, as the N-to-S acyl shift device. The selenoester selectively reacted with the terminal amino group of the peptide aryl thioester in the presence of N,N-diisopropylethylamine and dipyridyldisulfide, thus leaving the aryl thioester intact. Combined with silver-ion-promoted and silver-ion-free thioester activation methods, a one-pot four-segment ligation was realized. The method was successfully used to assemble the entire sequence of superoxide dismutase (SOD), which is composed of 153 amino-acid residues, in one pot. After the folding reaction, the fully active SOD was obtained.


Assuntos
Ésteres/metabolismo , Compostos Organosselênicos/metabolismo , Peptídeos/metabolismo , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/metabolismo , Ésteres/química , Estrutura Molecular , Compostos Organosselênicos/química , Peptídeos/química , Compostos de Sulfidrila/química , Superóxido Dismutase/química
17.
FEBS J ; 284(20): 3455-3469, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28834260

RESUMO

DNA methylation in promoter regions represses gene expression and is copied over mitotic divisions by Dnmt1. Dnmt1 activity is regulated by its replication foci targeting sequence (RFTS) domain which masks the catalytic pocket. It has been shown that Dnmt1 activity on unmethylated DNA is inhibited in nucleosome cores. In the present study, we aimed to assess the effect of nuclesome formation on maintenance methylation at single CpG resolution. We show that Dnmt1 fully methylates naked linker DNA in dinucleosomes, whereas maintenance methylation was repressed at all CpG sites in nucleosome core particles. Deletion of RFTS partly released obstruction of Dnmt1 activity in core particles. Histone H3 tail peptides inhibited Dnmt1 in an RFTS-dependent manner and repression was modulated by acetylation or methylation. We propose a novel function of RFTS to regulate Dnmt1 activity in nucleosomes.


Assuntos
Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Replicação do DNA , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Humanos , Deleção de Sequência
18.
Sci Rep ; 7(1): 8536, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819198

RESUMO

The chemokine receptor CCR7 contributes to various physiological and pathological processes including T cell maturation, T cell migration from the blood into secondary lymphoid tissues, and tumor cell metastasis to lymph nodes. Although a previous study suggested that the efficacy of CCR7 ligand-dependent T cell migration correlates with CCR7 homo- and heterodimer formation, the exact extent of contribution of the CCR7 dimerization remains unclear. Here, by inducing or disrupting CCR7 dimers, we demonstrated a direct contribution of CCR7 homodimerization to CCR7-dependent cell migration and signaling. Induction of stable CCR7 homodimerization resulted in enhanced CCR7-dependent cell migration and CCL19 binding, whereas induction of CXCR4/CCR7 heterodimerization did not. In contrast, dissociation of CCR7 homodimerization by a novel CCR7-derived synthetic peptide attenuated CCR7-dependent cell migration, ligand-dependent CCR7 internalization, ligand-induced actin rearrangement, and Akt and Erk signaling in CCR7-expressing cells. Our study indicates that CCR7 homodimerization critically regulates CCR7 ligand-dependent cell migration and intracellular signaling in multiple cell types.


Assuntos
Movimento Celular/fisiologia , Multimerização Proteica , Receptores CCR7/química , Transdução de Sinais , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocina CCL19/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Ligantes , Peptídeos/farmacologia , Ligação Proteica , Receptores CCR7/genética , Receptores CCR7/metabolismo
19.
Org Lett ; 19(14): 3771-3774, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28696114

RESUMO

The total synthesis of plusbacin A3 (1) has been accomplished using a solvent-dependent diastereodivergent Joullié-Ugi three-component reaction (JU-3CR) as a key step. Two trans-3-hydroxy-l-proline residues were constructed by combining the JU-3CR with a convertible isocyanide strategy. Subsequent peptide coupling and macrolactamization afforded plusbacin A3. Investigating the antibacterial activity of 1 compared with that of its dideoxy analogue revealed that the threo-ß-hydroxyaspartic acid residues are essential for antibacterial activity. Notably, there is a low potential for the development of resistance in S. aureus against plusbacin A3.

20.
Angew Chem Int Ed Engl ; 56(20): 5522-5526, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28394477

RESUMO

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7UA ,C7UB ] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A- and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (τ1/2 ≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.


Assuntos
Insulina/química , Insulina/síntese química , Sequência de Aminoácidos , Cristalografia por Raios X , Dissulfetos/química , Insulina/análogos & derivados , Modelos Moleculares
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