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1.
Peptide C‐terminal N‐alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C‐terminal N‐alkyl amides using the well‐known Fukuyama N‐alkylation reaction on a standard resin commonly used for the synthesis of peptide C‐terminal primary amides, the peptide amide linker‐polyethylene glycol‐polystyrene (PAL‐PEG‐PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N‐alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N‐alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N‐alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N‐alkyl amides. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 715–722, 2011. 相似文献
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Dong Yan GuangYu An Macus Tien Kuo 《Journal of cellular and molecular medicine》2016,20(11):2013-2019
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Two aspects were studied to elucidate the functional and structural characterization of apidaecin and its N-terminal and C-terminal fragments: (i) Functions of the N-terminal and C-terminal fragments of apidaecin were first studied by measuring their antibacterial activity, their ability to enter Escherichia coli cells and their effects on the activities of beta-galactosidase and alkaline phosphatase. The results indicate that neither the N-terminal nor the C-terminal of apidaecin contains intracellular delivery unit or active segment. (ii) The effect of apidaecin on the ATPase activity of DnaK, and the interactions of apidaecin with E.coli lidless DnaK and DnaK D-E helix were studied. Results showed that apidaecin could interact with the E.coli lidless DnaK protein and stimulate its ATPase activity, but not with E.coli DnaK D-E helix. This indicated that the antimicrobial activity of apidaecin may be shown by stimulating the ATPase activity of DnaK by binding to its conventional substrate-binding site, to decrease its cellular concentration of DnaK by competing with natural substrates and inhibit the enzymes' activities of E. coli cells. It is the first study to suggest that the apidaecin-binding site of DnaK is the conventional substrate binging site. 相似文献
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WEHI-231 B lymphoma cells have been employed for analysis of antigen-induced B cell unresponsiveness because these cells undergo cell cycle arrest in G1, accompanied by induction of apoptosis. In the present study, we examined the requirement for toxic small molecules apoptosis-inducing factor (AIF) and cytochrome c, and subsequent caspase activation in apoptotic cell death in WEHI-231 and CH31 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Pan-caspase inhibitor BD-fmk blocked mIg-mediated increase in cells with sub-G1 DNA content, whereas it did not affect mIg-mediated loss of mitochondrial membrane potential and phosphatidylserine exposure on B cell membrane. Dominant-negative form of c-Jun NH2-terminal kinase1 (JNK1) blocked the translocation of AIF into the nuclei and cytosol from the mitochondria in the WEHI-231 and CH31 cells following mIg engagement, whereas constitutively active form of JNK1 enhanced it. This AIF translocation was also blocked by Bcl-xL, but not by BD-fmk. Moreover, AIF-deficient clones via small interfering RNA (siRNA)-mediated method showed small increase in loss of mitochondrial membrane potential. After mIg engagement, the AIF-deficient clones displayed an enhanced sensitivity to mIg-mediated apoptosis, concomitant with translocation of a residual AIF into the nuclei, compared with control clone. Our findings are compatible with the notion that AIF has dual role, with a proapoptotic function in the nuclei and a distinct anti-apoptotic function in the mitochondria. These observations would be valuable for analysis of B cell unresponsiveness and hopefully for treatment of diseases involving B cell dysfunction. 相似文献
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Sonia Fieulaine Michel Desmadril Thierry Meinnel Carmela Giglione 《Acta Crystallographica. Section D, Structural Biology》2014,70(2):242-252
Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of the N‐formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three‐dimensional structure, are functionally interchangeable in vivo and display similar properties in vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate‐binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human‐like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand‐binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate. 相似文献
6.
Yu‐xi Zhang Chui‐ze Kong Lin‐hui Wang Jin‐yi Li Xian‐kui Liu Bin Xu Chuan‐liang Xu Ying‐hao Sun 《Journal of cellular biochemistry》2010,109(4):764-773
Androgen‐independent prostate cancers express high levels of Bcl‐2, and this over‐expression of Bcl‐2 protects prostate cancer cells from undergoing apoptosis. Ursolic acid (UA) has demonstrated an anti‐proliferative effect in various tumor types. The aim of this study is to evaluate the difference between UA‐induced apoptosis in androgen‐dependent prostate cancer cell line LNCaP cells and androgen‐independent prostate cancer cell line LNCaP‐AI cells and to reveal the molecular mechanisms underlying the apoptosis. We found that UA treatment in vitro can effectively induce apoptosis in LNCaP and LNCaP‐AI cells. UA can overcome Bcl‐2‐mediated resistance to apoptosis in LNCaP‐AI cells. Intrinsic apoptotic pathways can be triggered by UA treatment because c‐Jun N‐terminal kinase (JNK) is activated and subsequently provokes Bcl‐2 phosphorylation and degradation, inducing activation of caspase‐9. Although further evaluation is clearly needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer. J. Cell. Biochem. 109: 764–773, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
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Baisong Zheng Wen Yang Yuguo Wang Zhiyong Lou Yan Feng 《Acta Crystallographica. Section F, Structural Biology Communications》2011,67(10):1218-1220
It is well known that protein cocrystallization is affected by several parameters such as the ratio of the protein to the ligand, the reservoir solution, the pH and the temperature. Previously, spatial blocking by the N‐terminus was observed in the active site in the crystal structure of the native protein of a thermostable endoglucanase from the thermophilic bacterium Fervidobacterium nodosum Rt17‐B1 (FnCel5A). It was speculated that the N‐terminal α‐helix might form interactions with the substrate‐binding residues and it was believed that this spatial block is special to some extent. In order to confirm the effect on cocrystallization, two N‐terminally truncated variants of FnCel5A were constructed, purified and cocrystallized at 291 K. A crystal of FnCel5AND_12–343 in complex with cellobiose was obtained using PEG 8000 as a precipitant. A 2.2 Å resolution data set was collected. This crystal form (space group P41212, unit‐cell parameters a = b = 47.3, c = 271.4 Å) differed from that of the native protein. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.05 Å3 Da−1. 相似文献
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Five bombesin analogs with different functional groups at the C-terminus were synthesized using a solid-phase strategy. The protocols were optimized using 4-(hydroxymethyl)benzoic acid (HMBA) resin to synthesize a common precursor followed by nucleophilic cleavage of the base sensitive peptide ester linkage. The C-terminal modifications included ethylamide, butylamide, methyl ester, propyl ester and hydrazide. Cleavage from the resin was possible with the fully protected or deprotected precursor peptide; however, higher purity of the final products was achieved when cleavage protocols were conducted after side-chain deprotection. The synthesized peptides were analyzed and characterized using reverse phase HPLC and ESI-MS. The peptides were obtained in 13-32% overall recovery, calculated from the coupling efficiency of the first amino acid residue, and in 91-97% purity. 相似文献
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Ana Lucia L. R. Zimbardi Matheus P. Pinheiro Marcelo Dias‐Baruffi M. Cristina Nonato 《Acta Crystallographica. Section F, Structural Biology Communications》2010,66(5):542-545
Galectin‐4 is a tandem‐repeat‐type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin‐4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate‐binding specificities of galectin‐4 has been focused on. As an initial step, the N‐terminal carbohydrate‐recognition domain of human galectin‐4 (hGal4‐CRD‐1) has been successfully crystallized using the vapour‐diffusion technique, a complete data set has been collected to 2.2 Å resolution and the structure has been solved by the molecular‐replacement technique. The crystals belonged to space group P6122, with unit‐cell parameters a = b = 71.25, c = 108.66 Å. The asymmetric unit contained one molecule of hGal4‐CRD‐1, with a VM value of 2.34 Å3 Da−1 and a solvent content of 47.51%. 相似文献
13.
Chronic elevation of NEFAs (non‐esterified fatty acids) due to insulin resistance and obesity has been shown to be associated with increased β‐cell apoptosis and with the aetiology of the reduced β‐cell mass of Type 2 diabetes. SAPK (stress‐activated protein kinase)/JNK (c‐Jun N‐terminal kinase) have been implicated in the control of apoptosis. C‐K [compound K; 20‐O‐β‐d ‐glucopyranosyl‐20(S)‐protopanaxadiol] is the main intestinal bacterial metabolite of protopanaxadiol ginsenosides. Currently, little is known about the effects of C‐K on β‐cells with the presence of NEFAs. The aim of the present study was to investigate the in vitro protective effect of C‐K on MIN6N8 mouse insulinoma β‐cells against NEFA‐induced apoptosis, as well as the modulating effect on SAPK/JNK activation. Our results have shown that C‐K inhibited the palmitate‐induced apoptosis through modulating SAPK/JNK activation. We conclude that C‐K protects against β‐cell death and that, by anti‐apoptotic activity, C‐K may contribute to the previously reported anti‐diabetic actions of ginseng. 相似文献
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乳铁素——来源于乳铁蛋白的多功能抗菌肽 总被引：2，自引：0，他引：2
乳铁素是乳铁蛋白在酸性环境条件下经胃蛋白酶水解从N-端释放的多功能活性多肽．乳铁素不仅保持了完整乳铁蛋白的大部分生物学活性,而且乳铁素的某些生物学活性比乳铁蛋白更强．乳铁素具有抗细菌、抗真菌、抗病毒、抗肿瘤、免疫调节和抗炎症等多种生物学功能．然而,乳铁素的生物学作用大部分是通过体外试验发现和验证的,乳铁素的体内生物学效应还需更多的试验加以评价和证实,现代基因组学和蛋白组学分析方法和技术将有助于深入了解乳铁素体内生物学作用机制．本文就乳铁素的结构、生物学功能及其作用机制、制备和应用前景作一综述． 相似文献
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Vassiliki A. Iconomidou Danae Pheida Eftihia S. Hamodraka Claude Antony Andreas Hoenger Stavros J. Hamodrakas 《Peptide Science》2012,98(1):67-75
Deposition of amyloid in the atria (isolated atrial/cardiac amyloid) is fairly common in the aging heart. It consists of amyloid fibrils, formed both by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and the precursor molecule of ANP, proANP. This study examines whether amyloidogenic determinants (short peptides/amyloid forming favoring regions) exist in the sequence of NT‐proBNP, the N‐terminal part of proBNP, and if these determinants form amyloid‐like fibrils in vitro. We have predicted a possible amyloidogenic determinant in the sequence of the NT‐proBNP, and we conclusively show, after its synthesis, that it forms amyloid‐like fibrils in vitro, utilizing transmission electron microscopy, X‐ray diffraction, attenuated total reflectance Fourier‐transform infrared spectroscopy, and polarizing microscopy. Thus, for the first time, in this study, a possible biological role is attributed to a certain, specific part of this important cardiac prohormone/natriuretic peptide, which acts as an important biomarker indicative of heart failure. Its possible direct involvement in isolated cardiac amyloidosis, atrial fibrillation, and other types of cardiac amyloidoses is indicated and discussed. Since these cardiac hormones and their prohormones play key roles in cardiovascular homeostasis through natriuresis, diuresis, vasorelaxation, and inhibition of renin and aldosterone secretion (pathophysiology of hypertension and cardiovascular regulation), we also try to suggest these specific, short peptides as possible future structural targets of efforts toward inhibiting formation of natriuretic peptide(s) amyloid. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 67–75, 2012. 相似文献
17.
6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model. 相似文献
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Kusum Vats Rohit Sharma Mythili Kameswaran Haladhar Dev Sarma Drishty Satpati Ashutosh Dash 《Journal of peptide science》2019,25(7)
The present study describes modification of asparagine–glycine–arginine (NGR) peptide at N‐terminally and C‐terminally by introduction of a tridentate chelating scaffold via click chemistry reaction. The N‐terminal and C‐terminal modified peptides were radiometalated with [99mTc(CO)3]+ precursor. The influence of these moieties at the two termini on the targeting properties of NGR peptide was determined by in vitro cell uptake studies and in vivo biodistribution studies. The two radiolabeled constructs did not exhibit any significant variation in uptake in murine melanoma B16F10 cells during in vitro studies. In vivo studies revealed nearly similar tumor uptake of N‐terminally modified peptide construct 5 and C‐terminally construct 6 at 2 h p.i. (1.9 ± 0.1 vs 2.4 ± 0.2% ID/g, respectively). The tumor‐to‐blood (T/B) and tumor‐to‐liver (T/L) ratios of the two radiometalated peptides were also quite similar. The two constructs cleared from all the major organs (heart, lungs, spleen, stomach, and blood) at 4 h p.i. (<1% ID/g). Blocking studies carried out by coinjection of cCNGRC peptide led to approximately 50% reduction in the tumor uptake at 2 h p.i. This work thus illustrates the possibility of convenient modification/radiometalation of NGR peptide at either N‐ or C‐terminus without hampering tumor targeting and pharmacokinetics. 相似文献
19.
Rongjin Guan Sriram Aiyer Marie L. Cote Rong Xiao Mei Jiang Thomas B. Acton Monica J. Roth Gaetano T. Montelione 《Proteins》2017,85(4):647-656
The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656. © 2016 Wiley Periodicals, Inc. 相似文献
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Caroline Proulx Falko Noë Stan Yoo Michael D. Connolly Ronald N. Zuckermann 《Peptide Science》2016,106(5):726-736
A novel approach to sequentially degrade peptoid N‐terminal N‐(substituted)glycine residues on the solid‐phase using very mild conditions is reported. This method relies on the treatment of resin‐bound, bromoacetylated peptoids with silver perchlorate in THF, leading to an intramolecular cyclization reaction to liberate the terminal residue as a N‐substituted morpholine‐2,5‐dione, resulting in a truncated peptoid upon hydrolysis and a silver bromide byproduct. Side‐chain functional group tolerance is explored and reaction kinetics are determined. In a series of pentapeptoids possessing variable, non‐nucleophilic side‐chains at the second position (R2), we demonstrate that sequential N‐terminal degradation of the first two residues proceeds in 87% and 74% conversions on average, respectively. We further demonstrate that the degradation reaction is selective for peptoids, and represents substantial progress toward a mild, iterative sequencing method for peptoid oligomers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 726–736, 2016. 相似文献