Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.     

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.     

钒、铬团簇的电子亲合能、硬度与原子数的关系

根据静电球形液滴模型理论和离子极化、屏蔽效应对团簇的影响, 推导适合钒、铬团簇电子亲合能、硬度与原子数关系符合的普遍公式: Y=ae2/R+b/R2+c, R=rsN1/3, 从而更方便地预测大尺寸团簇的性质参数, 并发现和分析结构和性质异常的小团簇, 如Vn(n=5, 7, 9, 13)和Crn(n=6, 10, 17)的性质.     

Kinetic Insights into the Elongation Reaction of Actin Filaments as a Function of Temperature,Pressure, and Macromolecular Crowding

Actin polymerization is an essential process in eukaryotic cells that provides a driving force for motility and mechanical resistance for cell shape. By using preformed gelsolin–actin nuclei and applying stopped‐flow methodology, we quantitatively studied the elongation kinetics of actin filaments as a function of temperature and pressure in the presence of synthetic and protein crowding agents. We show that the association of actin monomers to the pointed end of double‐stranded helical actin filaments (F‐actin) proceeds via a transition state that requires an activation energy of 56 kJ mol^?1 for conformational and hydration rearrangements, but exhibits a negligible activation volume, pointing to a compact transition state that is devoid of packing defects. Macromolecular crowding causes acceleration of the F‐actin elongation rate and counteracts the deteriorating effect of pressure. The results shed new light on the combined effect of these parameters on the polymerization process of actin, and help us understand the temperature and pressure sensitivity of actin polymerization under extreme conditions.     

Blocking of the PD‐1/PD‐L1 Interaction by a D‐Peptide Antagonist for Cancer Immunotherapy

Blockade of the protein–protein interaction between the transmembrane protein programmed cell death protein 1 (PD‐1) and its ligand PD‐L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror‐image phage display, we developed the first hydrolysis‐resistant D ‐peptide antagonists to target the PD‐1/PD‐L1 pathway. The optimized compound ^DPPA‐1 could bind PD‐L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor‐bearing mice experiments indicated that ^DPPA‐1 could also effectively disrupt the PD‐1/PD‐L1 interaction in vivo. Thus D ‐peptide antagonists may provide novel low‐molecular‐weight drug candidates for cancer immunotherapy.     

Insights into the Dynamics and Binding of Two Polyprotein Substrate Cleavage Points in the Context of the SARS-CoV-2 Main and Papain-like Proteases

It is well known that vital enzymes in the replication process of the coronavirus are the SARS-CoV-2 PLpro and SARS-CoV-2 3CLpro, both of which are important targets in the search for anti-coronavirus agents. These two enzymes are responsible for cleavage at various polyprotein sites in the SARS-CoV-2 lifecycle. Herein, the dynamics of the polyprotein cleavage sequences for the boundary between non-structural proteins Nsp1 and Nsp2 (CS1) and between Nsp2 and Nsp3 (CS2) in complex with both the papain-like protein PLpro and the main protease 3CLpro were explored using computational methods. The post dynamics analysis reveals that CS1 and CS2 both have greater stability when complexed with PLpro. Of these two, greater stability is observed for the CS1–PLpro complex, while destabilization resulting in loss of CS2 from the PLpro active site is observed for CS2-PLpro, suggesting the rate of exchange by the papain-like protease is faster for CS2 compared to CS1. On the other hand, the 3CLpro main protease also reveals stability for CS1 suggesting that the main protease could also play a potential role in the cleavage at point CS1. However, destabilization occurs early in the simulation for the complex CLpro–CS2 suggesting a poor interaction and non-plausible protease cleavage of the polyprotein at CS2 by the main protease. These findings could be used as a guide in the development and design of potent COVID-19 antiviral inhibitors that mimic the CS1 cleavage site.     

Metallodrug Profiling against SARS-CoV-2 Target Proteins Identifies Highly Potent Inhibitors of the S/ACE2 interaction and the Papain-like Protease PLpro

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has called for an urgent need for dedicated antiviral therapeutics. Metal complexes are commonly underrepresented in compound libraries that are used for screening in drug discovery campaigns, however, there is growing evidence for their role in medicinal chemistry. Based on previous results, we have selected more than 100 structurally diverse metal complexes for profiling as inhibitors of two relevant SARS-CoV-2 replication mechanisms, namely the interaction of the spike (S) protein with the ACE2 receptor and the papain-like protease PL^pro. In addition to many well-established types of mononuclear experimental metallodrugs, the pool of compounds tested was extended to approved metal-based therapeutics such as silver sulfadiazine and thiomersal, as well as polyoxometalates (POMs). Among the mononuclear metal complexes, only a small number of active inhibitors of the S/ACE2 interaction was identified, with titanocene dichloride as the only strong inhibitor. However, among the gold and silver containing complexes many turned out to be very potent inhibitors of PL^pro activity. Highly promising activity against both targets was noted for many POMs. Selected complexes were evaluated in antiviral SARS-CoV-2 assays confirming activity for gold complexes with N-heterocyclic carbene (NHC) or dithiocarbamato ligands, a silver NHC complex, titanocene dichloride as well as a POM compound. These studies might provide starting points for the design of metal-based SARS-CoV-2 antiviral agents.     

Marine derived compounds as binders of the White spot syndrome virus VP28 envelope protein: In silico insights from molecular dynamics and binding free energy calculations

White spot syndrome virus (WSSV) remains as one of the most dreadful pathogen of the shrimp aquaculture industry owing to its high virulence. The cumulative mortality reaches up to 100% within in 2–10 days in a shrimp farm. Currently, no chemotherapeutics are available to control WSSV. The viral envelope protein, VP28, located on the surface of the virus particle acts as a vital virulence factor in the initial phases of inherent WSSV infection in shrimp. Hence, inhibition of envelope protein VP28 could be a novel way to deal with infection by inhibiting its interaction in the endocytic pathway. In this direction, a timely attempt was made to recognize a potential drug candidate of marine origin against WSSV using VP28 as a target by employing in silico docking and molecular dynamic simulations. A virtual library of 388 marine bioactive compounds was extracted from reports published in Marine Drugs. The top ranking compounds from docking studies were chosen from the flexible docking based on the binding affinities (ΔGb). In addition, the MD simulation and binding free energy analysis were implemented to validate and capture intermolecular interactions. The results suggested that the two compounds obtained a negative binding free energy with −40.453 kJ/mol and −31.031 kJ/mol for compounds with IDs 30797199 and 144162 respectively. The RMSD curve indicated that 30797199 moves into the hydrophobic core, while the position of 144162 atoms changes abruptly during simulation and is mostly stabilized by water bridges. The shift in RMSD values of VP28 corresponding to ligand RMSD gives an insight into the ligand induced conformational changes in the protein. This study is first of its kind to elucidate the explicit binding of chemical inhibitor to WSSV major structural protein VP28.     

Stereochemical Determination of Tuscolid/Tuscorons and Total Synthesis of Tuscoron D and E: Insights into the Tuscolid/Tuscoron Rearrangement

The stereochemistry of the structurally unique myxobacterial polyketides tuscolid/tuscorons was determined by a combination of high‐field NMR studies, molecular modeling, and chemical derivatization and confirmed by a modular total synthesis of tuscorons D and E. Together with the discovery of three novel tuscorons, this study provides detailed insight into the chemically unprecedented tuscolid/tuscoron rearrangement cascade.     

Studies towards the Design and Synthesis of Novel 1,5-Diaryl-1H-imidazole-4-carboxylic Acids and 1,5-Diaryl-1H-imidazole-4-carbohydrazides as Host LEDGF/p75 and HIV-1 Integrase Interaction Inhibitors

Two targeted sets of novel 1,5-diaryl-1H-imidazole-4-carboxylic acids 10 and carbohydrazides 11 were designed and synthesized from their corresponding ester intermediates 17, which were prepared via cycloaddition of ethyl isocyanoacetate 16 and diarylimidoyl chlorides 15. Evaluation of these new target scaffolds in the AlphaScreen^TM HIV-1 IN-LEDGF/p75 inhibition assay identified seventeen compounds exceeding the pre-defined 50% inhibitory threshold at 100 µM concentration. Further evaluation of these compounds in the HIV-1 IN strand transfer assay at 100 μM showed that none of the compounds (with the exception of 10a, 10l, and 11k, with marginal inhibitory percentages) were actively bound to the active site, indicating that they are selectively binding to the LEDGF/p75-binding pocket. In a cell-based HIV-1 antiviral assay, compounds 11a, 11b, 11g, and 11h exhibited moderate antiviral percentage inhibition of 33–45% with cytotoxicity (CC₅₀) values of >200 µM, 158.4 µM, >200 µM, and 50.4 µM, respectively. The antiviral inhibitory activity displayed by 11h was attributed to its toxicity. Upon further validation of their ability to induce multimerization in a Western blot gel assay, compounds 11a, 11b, and 11h appeared to increase higher-order forms of IN.     

In Silico Insights into the Mechanism of Action of Epoxy-α-Lapachone and Epoxymethyl-Lawsone in Leishmania spp.

Epoxy-α-lapachone (Lap) and Epoxymethyl-lawsone (Law) are oxiranes derived from Lapachol and have been shown to be promising drugs for Leishmaniases treatment. Although, it is known the action spectrum of both compounds affect the Leishmania spp. multiplication, there are gaps in the molecular binding details of target enzymes related to the parasite’s physiology. Molecular docking assays simulations were performed using DockThor server to predict the preferred orientation of both compounds to form stable complexes with key enzymes of metabolic pathway, electron transport chain, and lipids metabolism of Leishmania spp. This study showed the hit rates of both compounds interacting with lanosterol C-14 demethylase (−8.4 kcal/mol to −7.4 kcal/mol), cytochrome c (−10.2 kcal/mol to −8.8 kcal/mol), and glyceraldehyde-3-phosphate dehydrogenase (−8.5 kcal/mol to −7.5 kcal/mol) according to Leishmania spp. and assessed compounds. The set of molecular evidence reinforces the potential of both compounds as multi-target drugs for interrupt the network interactions between parasite enzymes, which can lead to a better efficacy of drugs for the treatment of leishmaniases.     

Insights into the surface property and blood compatibility of polyethersulfone/polyvinylpyrrolidone composite membranes: toward high‐performance hemodialyzer

Applications of blood purification membranes are fuelled by diverse clinical needs, such as hemodialysis, hemodiafiltration, hemofiltration, plasmapheresis, and plasma collection. For clinical usage, the adding of polyvinylpyrrolidone (PVP) is the general protocol for the design of antifouling and antithrombotic properties integrated artificial membranes. In the present work, to insight into the detailed surface properties and blood compatibilities of the PVP blended composite membranes, we synthesized a series of PVP polymers with different molecular weights using reversible addition fragmentation chain transfer polymerization and designed a series of polyethersulfone (PES)/PVP composite membranes by a physically blending method. The effects of PVP molecular weights and blending ratios on the surface properties and the blood compatibilities of the composite membranes were investigated in detail. The surface attenuated total reflection Fourier transform infrared spectra and scanning electron microscopy pictures indicated that the PVP was successfully immobilized into the membranes, and the composite membranes exhibited morphology transformation from finger‐like structure to sponge‐like structure, which indicated that the composite membrane had tunable porosity and permeability by adding PVP. The blood compatible tests revealed that the composite membranes showed increased hydrophilicity, decreased plasma protein adsorption, suppressed platelet adhesion, and prolonged blood clotting time compared with pristine PES membrane. These results indicated that the PES/PVP composite membranes exhibited enhanced antifouling and antithrombotic properties than the pristine PES membrane. Meanwhile, the results also suggested that the composite membranes with larger molecular weight PVP and higher blending ratios might show better blood compatibility. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation of the structure and properties of the potentially tautomeric 5,7-dimethyl-6H-pyrrolo[3,4-d]pyridazines in the gas and aqueous phases using the AM1 and PM3/COSMO solvation method

The potentially tautomeric 5,7-dimethyl-6H-pyrrolo[3,4-d]pyidazines, 2H and 6H, and their fixed tautomeric forms were studied in order to predict the most stable form by the restricted Hartree-Foch approach using semiempirical PM3 and AM1 quantum chemical calculations at the SCF level in the gas and aqueous phases. Both methods predicted that the 6H form, 5, is more stable than the other forms, 1-4, in both gas and aqueous phases. The results obtained were found to be in agreement with the experimental data. Monoprotonated forms of 5,7-dimethyl-6H-pyrrolo[3,4-d]pyridazines were also examined. Proton affinity calculations predicted that the first protonations take place on the N6 atom in the 2H form and on the N2 atom in the 6H form, resulting in a common cation 13.     

Multimolecular self-organization of acetylene and arylamines into 1-aryl-3-ethyl-4-vinylpyrroles in the KOBut/DMSO system

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稀土化合物TbCl3对成骨细胞系MC3T3-E1增殖、分化和矿化功能的影响

在细胞和分子水平上,研究了稀土化合物氯化铽(TbCl_3)对成骨细胞MC3T3-E1增殖、分化及矿化功能的影响。结果表明,细胞水平上,浓度为0.000 1、0.001、0.01、0.1、1和10μmol·L-1的TbCl_3均促进MC3T3-E1细胞的增殖、分化及其矿化功能,然而,当浓度升至为100和1 000μmol·L-1时,TbCl_3表现出抑制作用。分子水平上,浓度为0.000 1和0.1μmol·L-1的TbCl_3明显上调成骨分化相关基因骨形成蛋白2(BMP-2),碱性磷酸酶(ALP),骨涎蛋白(BSP),Ⅰ型胶原蛋白(ColⅠ),骨钙素(OCN)和runt相关转录因子2(Runx2)的表达。浓度为1 000μmol·L-1的TbCl_3则抑制上述成骨分化相关基因的表达。浓度为0.000 1、0.1和1μmol·L-1的TbCl_3促进成骨分化相关蛋白Runx2,BMP-2和OCN的表达;结果显示,低浓度的TbCl_3促进MC3T3-E1细胞的成骨分化及矿化功能,而高浓度TbCl_3则呈现出抑制作用。TbCl_3通过调控Runx2的表达刺激早期成骨分化相关基因BMP-2、ColⅠ和晚期成骨分化相关基因ALP、OCN的表达,从而诱导MC3T3-E1成骨分化。