Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC‐associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA‐based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC‐associated proteins. This study provided a simple and broadly applicable method for characterizing protein‐protein interactions.     

Identification of Proteins Using Affinity Affi-Gel Simultaneously with Acrylamide Electrophoresis

Abstract

This work presents a method in which a desired protein may be separated from a biological protein preparation and identified by highly specific monoclonal antibody. The desired protein may be isolated from crude protein preparations by this method. The protein remains intact and stable throughout the procedure. This method utilizes common nonreducing conditions of polyacrylamide gel electrophoresis (PAGE), and Western Blot transfer of protein. This isolation of the desired protein can be accomplished for a microgram or tens of micrograms of material. The application of Western blot technique with monoclonal antibody permits the highest specificity in terms of identifying the target protein. In addition to identifying the target protein the method can isolate sufficient amounts for further characterization.     

新型吡啶苯磺酰胺联喹啉基异羟肟酸类PI3K/HDAC双靶点抑制剂的设计、合成和生物活性研究

多靶点药物已成为一种有广阔前景的药物,特别是对抗肿瘤药物的研发.基于候选药物GSK2126458和上市药物Vorinostat的结构特点,设计并合成了一系列新型的磷脂酰肌醇3-激酶(PI3Ks)和组蛋白脱乙酰酶(HDACs)双重抑制剂.生物活性研究发现,化合物GYB-4对PI3Kα和HDAC1的IC50分别为1.0和4.2nmol/L;化合物GYB-5对PI3Kα和HDAC1的IC50分别为1.3和4.8nmol/L.对所有化合物在HCT116,PC3和A2780细胞株上进行了增殖抑制活性研究,相关的构效关系研究将为PI3K和HDAC双靶点抑制剂的进一步优化提供思路.     

Insight into the Complexation Mode of Bis(nitrilotriacetic acid) (NTA) Ligands with Ni2+ Involved in the Labeling of Histidine‐Tagged Proteins

According to literature reports and our own findings, the binding of new Ni²⁺‐preloaded bis(nitrilotriacetic acid) (NTA) ligands with polyhistidine‐tagged proteins has been found to be accompanied by a one‐ to two‐order‐of‐magnitude increase in affinity, compared to the binding of a single Ni²⁺‐preloaded NTA moiety. In spite of the introduction of a second NTA chelating group, a cooperative effect that is less than the theoretical maximum has been observed. Herein, we present a rational explanation for the observed stability of the ternary complex involving the postulated bis‐NTA–(Ni²⁺)₂ species and multivalent polyhistidine tags. We have found that prior to the formation of the ternary complex, the Ni²⁺‐preloading step of bis‐NTA ligands does not form the expected bis‐NTA–(Ni²⁺)₂ exclusively. Instead of the major formation of bis‐NTA–(Ni²⁺)₂ species, it appears that cyclic discrete 1:1 and 2:2 entities are predominantly formed. It is proposed that these species interact upon ring‐opening with multivalent histidine tags. The occurrence of this phenomena accounts for the overall one‐ to two‐order‐of‐magnitude increase in affinity of ternary complexes involving bis‐NTA ligands.     

Site‐Specific Labeling of Proteins with a Chemically Stable,High‐Affinity Tag for Protein Study

Site‐specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biology. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure determination and mobility studies of proteins and protein–ligand complexes. Herein, we present a new way to label proteins in a site‐specific manner with a high‐affinity and chemically stable tag, 4‐vinyl(pyridine‐2,6‐diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: 1) it has a very high binding affinity for lanthanides (higher than EDTA), 2) there is no heterogeneity in complexes with lanthanides, 3) the derivatized protein is stable and potentially applicable to the in situ analysis of proteins.     

蛋白A高效亲和膜色谱法测定人血浆中免疫球蛋白G的含量

研究了蛋白Ａ高效亲和膜色谱对水溶液及人血浆中人免疫球蛋白Ｇ（ＨＩｇＧ）的特异性吸附和定量测定。方法具有较高的精确度和较好的重复性：ＨＩｇＧ标样５次重复进样的相对标准偏差为１．５％，人血浆样品３次重复进样的相对标准偏差为３．６％；所测得的定量标定曲线的线性相关系数达到０．９９９３；不含己二胺间隔臂的亲和介质的非特异性吸附极低，基本检测不出来。快速实验中，一次分析可在０．５ｍｉｎ内完成。实验表明，利用所建立的方法对人血浆中的ＨＩｇＧ进行定量测定可以得到较为满意的结果。     

Preparation of Immobilized Metal Affinity Chromatographic Packings by Immobilization of Carboxymethylated Asparate (CM-Asp) Based on Monodisperse Hydrophilic Non-porous Beads and Their Application

The hydrophilic immobilized metal affinity chromatographic packing was prepared by immobilization of carboxymethylated asparate (CM‐Asp) as chelating ligand and Ni²⁺ as center ion on the base of monodispersed, 3.0 µm non‐porous monodisperse poly(glycidylmethacrylate‐co‐ethylenedimethacrylate) (P_GMA/EDMA) particles. The retention behavior of proteins and the effect of pH on the retention in the range from 4.0 to 9.0 were investigated on both the naked and metal ion chelated columns. Four proteins were quickly separated in 2.0 min with linear gradient elution at a flow rate of 3.0 mL·min^?1 by using the synthesized Ni²⁺‐CM‐Asp‐P_GMA/EDMA packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. The Ni²⁺‐CM‐Asp‐P_GMA/EDMA column was further investigated for the rapid separation and purification of copper‐zinc superoxide dismutase (Cu,Zn‐SOD) from the blood of pig in 3.0 min with only one step. The results obtained were satisfactory.     

Quantitative FRET (qFRET) Technology for the Determination of Protein–Protein Interaction Affinity in Solution

Protein–protein interactions play pivotal roles in life, and the protein interaction affinity confers specific protein interaction events in physiology or pathology. Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research to detect molecular interactions in vitro and in vivo. The FRET assay provides very high sensitivity and efficiency. Several attempts have been made to develop the FRET assay into a quantitative measurement for protein–protein interaction affinity in the past. However, the progress has been slow due to complicated procedures or because of challenges in differentiating the FRET signal from other direct emission signals from donor and receptor. This review focuses on recent developments of the quantitative FRET analysis and its application in the determination of protein–protein interaction affinity (K_D), either through FRET acceptor emission or donor quenching methods. This paper mainly reviews novel theatrical developments and experimental procedures rather than specific experimental results. The FRET-based approach for protein interaction affinity determination provides several advantages, including high sensitivity, high accuracy, low cost, and high-throughput assay. The FRET-based methodology holds excellent potential for those difficult-to-be expressed proteins and for protein interactions in living cells.     

Poly(hydroxyether of phenolphthalein) and its blends with poly(ethylene oxide)

Poly(hydroxyether of phenolphthalein) (PPH) was synthesized through the polycondensation of phenolphthalein with epichlorohydrin. It was characterized by Fourier transform infrared (FTIR) spectroscopy, NMR spectroscopy, and differential scanning calorimetry (DSC). The miscibility of the blends of PPH with poly(ethylene oxide) (PEO) was established on the basis of the thermal analysis results. DSC showed that the PPH/PEO blends prepared via casting from N,N‐dimethylformamide possessed single, composition‐dependent glass‐transition temperatures. Therefore, the blends were miscible in the amorphous state for all compositions. FTIR studies indicated that there were competitive hydrogen‐bonding interactions with the addition of PEO to the system, which were involved with OH…O?C〈, ? OH…? OH, and ? OH vs ether oxygen atoms of PEO hydrogen bonding, that is both intramolecular and intermolecular, between PPH and PEO). Some of the hydroxyl stretching vibration bands significantly shifted to higher frequencies, whereas others shifted to lower frequencies, and this suggested the formation of hydrogen bonds between the pendant hydroxyls of PPH and ether oxygen atoms of PEO, which were stronger than the intramolecular hydrogen bonding between hydroxyls and carbonyls of PPH. The FTIR spectra in the range of carbonyl stretching vibrations showed that the hydroxyl‐associated carbonyl groups were partially set free because of the presence of the competitive hydrogen‐bonding interactions. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 466–475, 2003     

Bringing the Science of Proteins into the Realm of Organic Chemistry: Total Chemical Synthesis of SEP (Synthetic Erythropoiesis Protein)

Erythropoietin, commonly known as EPO, is a glycoprotein hormone that stimulates the production of red blood cells. Recombinant EPO has been described as “arguably the most successful drug spawned by the revolution in recombinant DNA technology”. Recently, the EPO glycoprotein molecule has re‐emerged as a major target of synthetic organic chemistry. In this article I will give an account of an important body of earlier work on the chemical synthesis of a designed EPO analogue that had full biological activity and improved pharmacokinetic properties. The design and synthesis of this “synthetic erythropoiesis protein” was ahead of its time, but has gained new relevance in recent months. Here I will document the story of one of the major accomplishments of synthetic chemistry in a more complete way than is possible in the primary literature, and put the work in its contemporaneous context.     

Total Synthesis of the Bicyclic Depsipeptide HDAC Inhibitors Spiruchostatins A and B, 5′′‐epi‐Spiruchostatin B,FK228 (FR901228) and Preliminary Evaluation of Their Biological Activity

The bicyclic depsipeptide histone deacetylase (HDAC) inhibitors spiruchostatins A and B, 5′′‐epi‐spiruchostatin B and FK228 were efficiently synthesized in a convergent and unified manner. The synthetic method involved the following crucial steps: i) a Julia–Kocienski olefination of a 1,3‐propanediol‐derived sulfone and a L ‐ or D ‐malic acid‐derived aldehyde to access the most synthetically challenging unit, (3S or 3R,4E)‐3‐hydroxy‐7‐mercaptohept‐4‐enoic acid, present in a D ‐alanine‐ or D ‐valine‐containing segment; ii) a condensation of a D ‐valine‐D ‐cysteine‐ or D ‐allo‐isoleucine‐D ‐cysteine‐containing segment with a D ‐alanine‐ or D ‐valine‐containing segment to directly assemble the corresponding seco‐acids; and iii) a macrocyclization of a seco‐acid using the Shiina method or the Mitsunobu method to construct the requisite 15‐ or 16‐membered macrolactone. The present synthesis has established the C5′′ stereochemistry of spiruchostatin B. In addition, HDAC inhibitory assay and the cell‐growth inhibition analysis of the synthesized depsipeptides determined the order of their potency and revealed some novel aspects of structure–activity relationships. It was also found that unnatural 5′′‐epi‐spiruchostatin B shows extremely high selectivity (ca. 1600‐fold) for class I HDAC1 (IC₅₀=2.4 nM ) over class II HDAC6 (IC₅₀=3900 nM ) with potent cell‐growth‐inhibitory activity at nanomolar levels of IC₅₀ values.