Palmitoyl acyltransferases,their substrates,and novel assays to connect them (Review)

Thio-palmitoylation is the post-translational addition of the 16-carbon fatty acid, palmitate, to the thiol side chain of cysteine residues by a labile thioester bond. Palmitoylation increases the lipophilicity of a protein resulting in dramatic changes in its subcellular distribution such as moving from the endoplasmic reticulum to the plasma membrane or in subtle changes like an increased affinity for cholesterol-rich lipid rafts in membranes. Palmitoylation is also dynamic, making it unique among post-translational protein lipid modifications. Discovering the molecular identity of palmitoyl acyltransferases (PATs) was a watershed event that dramatically accelerated the pace of discovery in the field. Likewise, there has been increased interest in palmitoylation partly because many of the genes encoding PATs have been linked to cancer and other diseases. Now, with a greater understanding of how palmitate is enzymatically attached to proteins, some of the most interesting questions include: What are the substrates of each PAT?; how does a PAT recognize and palmitoylate a substrate?; how are PATs regulated?; and, how is depalmitoylation regulated? The answers to these questions are beginning to unfold due to the recent development of novel assays as well as the expansion and refinement of existing assays. Our ability to understand palmitoylation and its importance to human health and disease is only as good as the methods we use to test our hypotheses. The continued development of methods with increased sensitivity and selectivity is critical to this venture.     

Structure–activity studies on the upstream splice junction of a semisynthetic intein

Protein trans-splicing by split inteins holds great potential for the chemical modification and semisynthesis of proteins. However, the structural requirements of the extein sequences immediately flanking the intein are only poorly understood. This knowledge is of particular importance for protein labeling, when synthetic moieties are to be attached to the protein of interest as seamlessly as possible. Using the semisynthetic Ssp DnaB intein both in form of its wild-type sequence and its evolved M86 mutant, we systematically varied the sequence upstream of the short synthetic Int^N fragment using both proteinogenic amino acids and unnatural building blocks. We could show for the wild-type variant that the native N-extein sequence could be reduced to the glycine residue at the (?1) position directly flanking the intein without significant loss of activity. The glycine at this position is strongly preferred over building blocks containing a phenyl group or extended alkyl chain adjacent to the scissile amide bond of the N-terminal splice junction. Despite their negative effects on the splicing yields, these unnatural substrates were well processed in the N–S acyl shift to form the respective thioesters and did not result in an increased decoupling of the asparagine cyclization step at the C-terminal splicing junction. Therefore, the transesterification step appeared to be the bottleneck of the protein splicing pathway. The fluorophore 7-hydroxycoumarinyl-4-acetic acid as a minimal N-extein was efficiently ligated to the model protein, in particular with the M86 mutant, probably because of its higher resemblance to glycine with an aliphatic c-α carbon atom at the (?1) position. This finding indicates a way for the virtually traceless labeling of proteins without inserting extra flanking residues. Due to its overall higher activity, the M86 mutant appears most promising for many protein labeling and chemical modification schemes using the split intein approach.     

Insights from atomic-resolution X-ray structures of chemically synthesized HIV-1 protease in complex with inhibitors

The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.     

肾素(原)受体在大鼠肾小球系膜细胞和肾脏的表达

近年发现的肾素（原）受体（renin／prorenin receptor,RnR）已被证明具有生物学功能,在心、肾及多种细胞表达。本文旨在观察RnR在体外培养的大鼠肾小球系膜细胞（mesangial cells,MCs）和肾脏中是否表达,及其表达的细胞部位,并用RnR的多肽阻断剂肾素原“柄区肽”（handle region peptide,HRP）与RnR结合后观察受体复合物进入细胞的过程与定位。结果显示,RnR主要存在于大鼠肾脏皮质肾小球系膜区和体外培养的MCs的细胞核周围胞浆和细胞膜。将FITC标记的HRP（FITC-HRP）加入细胞培养液后30S到30min期间,可观察到FITC-HRP由培养液转移到胞浆内并进入细胞核。用免疫荧光和激光共聚焦技术观察到,HRP与RnR的共定位主要位于细胞膜和细胞核周围胞浆;在30min时,一部分HRP已进入细胞核,而RnR没有进入细胞核内,仍主要位于细胞核周围胞浆。上述结果提示,RnR与其配基结合后进入细胞内并发挥生物学效应。     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

The CC chemokine CCL14/HCC-1(9-74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9-39) and CCL14/HCC-1(40-74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9-39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

3-Thiopropionic acid as a highly versatile multidetachable thioester resin linker

Summary This paper describes a practical new use of 3-mercaptopropionic acid as a highly versatile multidetachable linker for solid-phase synthesis. Our approach is based on the stability of the alkylthioester functionality to optimized Boc-SPPS protocols and HF treatment, as well as on the mild activation of the thioester functionality toward nucleophilic or reductive displacement. This allows several C-terminal modifications to be introduced into a synthetic molecule during the cleavage step. We have shown that unprotected peptides can be efficiently cleaved from a propyl thioester-polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer resin using a great variety of nucleophiles to give the corresponding C-terminally modified peptides (esters, thioesters, carboxylic acids, thioacids, amides, hydroxamic acids, hydrazides, alcohols). The nucleophilic cleavage reaction is both rapid and exceptionally clean in all the cases tested. Abbreviations: HBTU,N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphateN-oxide); DIEA,N,N-diisopropylethylamine; DMF,N,N-dimethyl formamide; ES-MS, electrospray mass spectrometry; FAB-MS, fast atom bombardment mass spectrometry; HMBA, hydroxymethylbenzoic acid; HPLC, high performance liquid chromatography; PBS: phosphate buffer saline; PEGA, polyethylene glycolpoly-(N,N-dimethylacrylamide); TFA, trifluoroacetic acid; SPPS, solid-phase peptide synthesis. Standard IUPAC single and triple letter codes for amino acids are used throughout     

UBE1DC1, an ubiquitin-activating enzyme, activates two different ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins are known to be covalently conjugated to a variety of cellular substrates via a three-step enzymatic pathway. These modifications lead to the degradation of substrates or change its functional status. The ubiquitin-activating enzyme (E1) plays a key role in the first step of ubiquitination pathway to activate ubiquitin or ubiquitin-like proteins. Ubiquitin-activating enzyme E1-domain containing 1 (UBE1DC1) had been proved to activate an ubiquitin-like protein, ubiquitin-fold modifier 1 (Ufm1), by forming a high-energy thioester bond. In this report, UBE1DC1 is proved to activate another ubiquitin-like protein, SUMO2, besides Ufm1, both in vitro and in vivo by immunological analysis. It indicated that UBE1DC1 could activate two different ubiquitin-like proteins, SUMO2 and Ufm1, which have no significant similarity with each other. Subcellular localization in AD293 cells revealed that UBE1DC1 was especially distributed in the cytoplasm; whereas UBE1DC1 was mainly distributed in the nucleus when was cotransfected with SUMO2. It presumed that UBE1DC1 greatly activated SUMO2 in the nucleus or transferred activated-SUMO2 to nucleus after it conjugated SUMO2 in the cytoplasm.     

Structural characterization of CalO2: a putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic pathway

Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X-ray structure determination at 2.5 A resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2-catalyzed step may occur after CalO3-catalyzed iodination and may also require a coenzyme A- (CoA) or acyl carrier protein- (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well-ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures.     

Site-selective installation of an electrophilic handle on proteins for bioconjugation

Site-selective protein modification strategies can be used to insert non-natural functional groups into protein structures. Herein, we report on the use of the bis-electrophile 3-bromo-2-bromomethyl-1-propene as a reagent to introduce an electrophilic handle at cysteine residues under mild conditions. This method is demonstrated on a variety of proteins containing a solvent-exposed cysteine residue, including an anti-HER2 nanobody. Chemically distinct protein conjugates are then efficiently formed through further reaction of the electrophilic site with various nucleophiles, including thiols and amines. The resulting chemically-defined conjugates are highly stable in the presence of glutathione or human plasma and retain both the structure and function of the native protein.     

Advances in Fmoc solid‐phase peptide synthesis

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very‐high‐quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology. Improvements are being continually reported for peptide quality, synthesis time and novel synthetic targets. Topical peptide research has contributed to a continuous improvement and expansion of Fmoc SPPS applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.