Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptideproducing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1′ sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs. peptide ligase | data mining | ligase-activity determinant
Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na+, K+, Mg2+, Ca2+, cobalt-hexammine3+, spermidine3+ and spermine4+. Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K+, (as well as Rb+ and Cs+) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K+ (and Rb+/Cs+) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome–nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.
We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C"-thiocarboxylic acid reacting with a f8-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled in aqueous solution to give free peptides up to 54 residues long with high efficiency.Recent advances in the design of artificial proteins with unusual architectures (1) and the ready availability of large peptides and protein domains from both solid-phase synthesis (2, 3) and recombinant DNA have led to the need for the development of convergent strategies in peptide synthesis with these large unprotected peptide segments as building blocks. A requirement of such a strategy is the exceptionally high regiospecificity in the amide bond formation between the a-amine and Ca-acyl moiety of the two segments. Recently, we have proposed (4-7) an orthogonal coupling method to achieve this goal.An orthogonal coupling method to form an amide bond is conceptually different from an orthogonal protecting group strategy (8). It is site-specific and allows only a single specific coupling reaction between the C' moiety of one peptide segment and the Na-amine of another peptide segment, in the presence of other reactive amino moieties. In such a method, a capture step (9) brings the respective N and C termini into close proximity and the amide bond formation is effected by an intramolecular acyl transfer at high effective concentration. So far, all orthogonal couplings have exploited the unique reactivity of the 1,2-aminothiol moiety of an N-terminal cysteine on the amine segment to achieve selectivity to form thiazolidine, thioester, and disulfide with acyl segments bearing glycolaldehyde ester (4, 5), thioester (6, 10), and mixed disulfide (6, 7). In orthogonal couplings involving Ca-thioester and Na-cysteine, the capture step occurs when the covalent thioester is formed between these two segments leading to a spontaneous S-to N-acyl transfer to form a cysteinyl peptide bond (Fig. 1) using fully unprotected peptide segments. The capture is effected by a thiol-thioester exchange in method A and thioesterification in method B. Both will give a covalent thioester leading to a spontaneous intramolecular acyl transfer to give a cysteinyl peptide bond. P-I (shaded oval), peptide-1; P-2, peptide-2 (open square); R, CH2CH2CO2H.N-terminal ,3-bromoalanine (BrAla) and forms a similar covalent thioester that will rearrange rapidly via an intramolecular S-to-N acyl transfer to give the peptide bond (Fig. 1 Table 3), were synthesized by the Fmoc/t-Bu strategy on Wang resin (11) by using the benzotriazol-1-yl-oxy-t...
Summary Previous experiments suggest a connection between the N-alpha-acetylation of proteins and the sensitivity of cells to apoptotic signals. Here, we describe a novel biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the anti-apoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We conclude that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.
We describe the concept and the verification of a chemical ligation approach to the synthesis of proteins using peptide se-ents with no protecting groups and no activation of the C-tenal a-carboxyl group. This approach of three steps: (i) aldehyde introduction, in which a masked glycolaldehyde ester Is linked to the carboxyl terminus of an unprotected peptide by reverse protedlysis; (ii) ring formation, in which the umaked aldehyde reacts with the N-teinal a-amino group of the second unprotected peptide containing either a cysteine or a threonine residue to form a t ne or olidine ring at an acidic pH; and (iii) rearrangement in which O-acyl ester linkage is transferred to N-acyl amide linkage to form a peptide bond with a pseudoproline stuture at higher pH. The (4), and the semi-synthesis of proteins consisting of components from synthetic peptides with unusual amino acids and proteins obtained through recombinant technology.The conventional segment-synthesis approaches (5) usually require a maximal protection strategy which has the limitations ofpoor solubility, low coupling efficiency, and the possible danger of racemization due to the overactivation of the carboxyl group. Considerable efforts have been made to overcome these limitations through the use of partial or minimal protecting-group strategies and improvements in the activation methods (6-11). However, these approaches do not overcome the problem of the poor coupling efficiency between large peptide segments, because of the intrinsic difficulty of obtaining effective molar concentrations for high molecular weight molecules. Alternative approaches to overcome the difficulty of forming amide bonds use hydrazone (12,13) or thioether and thioester (14) as surrogate bonds. The advantage of these peptide-bond replacement approaches resides in the high and specific reactivity between the two functional groups not found in amino acids to make the conjugation efficient. The disadvantage is that these linkages are substantially different from the peptide bond, which may lead to conformational distortion, and are usually not stable at either acidic (12) or basic (14) pH.We have now designed and developed a chemical ligation method by which two unprotected peptide segments can be ligated together through an amide bond. In our design, we make use of a highly regiospecific and efficient reaction to link covalently two unprotected peptides. The selectivity of this reaction would also obviate the need for any protecting groups. A peptide bond is then formed in a second step through an intramolecular rearrangement between the two closely neighboring carboxyl and amino groups. The efficiency of the first reaction would solve the slow kinetic problem for reactions between large molecules, and the second reaction, designed as a spontaneous process to form amide bonds without activating the carboxyl group, would overcome the racemization problem of the conventional segment condensation method. EXPERIMENTAL PROCEDURESGeneral. 1H NMR spectra were obtained at 300 ...
ObjectiveTo perform a meta-analysis examining the efficacy of phytoestrogens for the relief of menopausal symptoms.MethodsMedline, Cochrane, EMBASE, and Google Scholar databases were searched until September 30, 2013 using the following key words: vasomotor symptoms, menopausal symptoms, phytoestrogens, isoflavones, coumestrol, soy, red clover. Inclusion criteria were (1) randomized controlled trial (RCT), (2) perimenopausal or postmenopausal women experiencing menopausal symptoms, (3) intervention with an oral phytoestrogen. Outcome measures included Kupperman index (KI) changes, daily hot flush frequency, and the likelihood of side-effects.ResultsOf 543 potentially relevant studies identified, 15 RCTs meeting the inclusion criteria were included. The mean age of the subjects ranged from 49 to 58.3 and 48 to 60.1 years, respectively, in the placebo and phytoestrogen groups. The number of participants ranged from 30 to 252, and the intervention periods ranged from 3 to 12 months. Meta-analysis of the seven studies that reported KI data indicated no significant treatment effect of phytoestrogen as compared to placebo (pooled mean difference = 6.44, p = 0.110). Meta-analysis of the ten studies that reported hot flush data indicated that phytoestrogens result in a significantly greater reduction in hot flush frequency compared to placebo (pooled mean difference = 0.89, p < 0.005). Meta-analysis of the five studies that reported side-effect data showed no significant difference between the two groups (p = 0.175).ConclusionPhytoestrogens appear to reduce the frequency of hot flushes in menopausal women, without serious side-effects.
A thiol group introduced on the gamma-carbon of lysine mediates robust native chemical ligation at both the alpha- and epsilon-amines in two consecutive steps. Desulfurization then affords the final product, in which the lysine residue at the ligation site has an isopeptide bond on its side chain. The method is useful for the synthesis of proteins containing special post-translational modifications on lysine.
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