Structural insights into the aggregation mechanism of huntingtin exon 1 protein fragment with different polyQ-lengths

Huntington disease is a neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) at the N-terminal of the huntingtin exon 1 protein. The detailed structure and the mechanism behind this aggregation remain unclear and it is assumed that the polyQ undergoes a conformational transition to the β-sheet structure when it aggregates. Investigating the misfolding of polyQ facilitates the determination of the molecular mechanism of aggregation and can potentially help in developing a novel approach to inhibit polyQ aggregation. Moreover, the flanking sequences of the polyQ region play a vital role in structural changes and the aggregation mechanism. We performed all-atom molecular dynamics simulations to gain structural insights into the aggregation mechanism using eight different models with glutamine repeat lengths Q₂₇, Q₂₇P₁₁, Q₃₄, Q₃₅, Q₃₆, Q₄₀, Q₅₀, and Q₅₀P₁₁. In the models without flanking polyPs, we noticed that the transformation of a random coil to β-sheet occurs when the number of Q increases. We also found that the flanking polyPs prevent aggregation by decreasing the probability of forming a β-sheet structure. When polyQ length increases, the 17 N-terminal flanking residues are more likely to adopt a β-sheet conformation from α-helix and coil. From our simulations, we suggest that at least 34 glutamines are required for initiating aggregation and 40 residues length is critical for the aggregation of huntingtin exon 1 protein for disease onset. This study provides structural insights into misfolding and the role of flanking sequences in huntingtin aggregation which will further help in developing therapeutic strategies for Huntington's disease.     

PLUG (Pruning of Local Unrealistic Geometries) removes restrictions on biophysical modeling for protein design

Protein design algorithms must search an enormous conformational space to identify favorable conformations. As a result, those that perform this search with guarantees of accuracy generally start with a conformational pruning step, such as dead-end elimination (DEE). However, the mathematical assumptions of DEE-based pruning algorithms have up to now severely restricted the biophysical model that can feasibly be used in protein design. To lift these restrictions, I propose to prune local unrealistic geometries (PLUG) using a linear programming-based method. PLUG's biophysical model consists only of well-known lower bounds on interatomic distances. PLUG is intended as preprocessing for energy-based protein design calculations, whose biophysical model need not support DEE pruning. Based on 96 test cases, PLUG is at least as effective at pruning as DEE for larger protein designs—the type that most require pruning. When combined with the LUTE protein design algorithm, PLUG greatly facilitates designs that account for continuous entropy, large multistate designs with continuous flexibility, and designs with extensive continuous backbone flexibility and advanced nonpairwise energy functions. Many of these designs are tractable only with PLUG, either for empirical reasons (LUTE's machine learning step achieves an accurate fit only after PLUG pruning), or for theoretical reasons (many energy functions are fundamentally incompatible with DEE).     

Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa

Type Three Secretion Systems (T3SS) from many gram-negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically, and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with V_max of 3.9 ± 0.2 μmol/min/mg and K _m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni-NTA pull-down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN–PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN–PscL heterotrimeric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN–PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN–PscL complex in T3SS mediated pathogenic pathway.     

Electrostatic interactions determine entrance/release order of substrates in the catalytic cycle of adenylate kinase

Adenylate kinase is a monomeric phosphotransferase with important biological function in regulating concentration of adenosine triphosphate (ATP) in cells, by transferring the terminal phosphate group from ATP to adenosine monophosphate (AMP) and forming two adenosine diphosphate (ADP) molecules. During this reaction, the kinase may undergo a large conformational transition, forming different states with its substrates. Although many structures of the protein are available, atomic details of the whole process remain unclear. In this article, we use both conventional molecular dynamics (MD) simulation and an enhanced sampling technique called parallel cascade selection MD simulation to explore different conformational states of the Escherichia coli adenylate kinase. Based on the simulation results, we propose a possible entrance/release order of substrates during the catalytic cycle. The substrate-free protein prefers an open conformation, but changes to a closed state once ATP·Mg enters into its binding pocket first and then AMP does. After the reaction of ATP transferring the terminal phosphate group to AMP, ADP·Mg and ADP are released sequentially, and finally the whole catalyze cycle is completed. Detailed contact and distance analysis reveals that the entrance/release order of substrates may be largely controlled by electrostatic interactions between the protein and the substrates.     

Association of Per3 length polymorphism with susceptibility of Alzheimer disease (AD) in Chinese population

Objectives: The association of Per3 length polymorphism with susceptibility of Alzheimer Disease (AD) was examined in the present study. Methods: This study was constructed using the case-control method and investigated the association of Per3 length polymorphism with susceptibility of AD. Genotypes of APOE and Per3 length were determined by a PCR restriction fragment length polymorphism detection method. Results: In this study, we gathered 130 unrelated AD patients and 188 controls in performing an analysis the association of Per3 length polymorphism with susceptibility of AD. In the whole sample or APOE ε4 non-carriers, an increased prevalence of five repeat homozygotes of Per3 length in AD patients had significant higher than that in controls (in the whole sample: χ² = 7.261,= 0.0176; in APOE ε4 non-carriers: χ² = 6.086, p = 0.030). And, among APOE ε4 carriers, an increased prevalence of five repeat homozygotes of Per3 length in AD patients had also significant higher than that in controls (χ² = 3.893, p = 0.0319). Conclusions: Among APOE ε4 non-carriers, five repeat homozygotes of Per3 length was associated with a high susceptibility of AD among APOE ε4 carriers and non-carriers.     

典型油气藏区域环境因素差异对甲烷氧化菌丰度与群落结构分布的影响

【目的】甲烷氧化菌(methane-oxidizing bacteria, MOB)是油气微生物勘探中重要的指标微生物,其丰度与群落结构分布受到地理位置和多种环境因素的影响。本研究以采集的7个典型油气藏区域的土壤样品为研究对象,探究油气藏区域环境因素差异对甲烷氧化菌丰度与群落结构分布的影响。【方法】对采集自7个典型油气藏区域土壤样品进行pmoA基因实时荧光定量PCR (real-time fluorescence quantitative PCR, qPCR)、细菌16S rRNA基因测序和甲烷氧化菌功能基因pmoA测序,结合环境因子,比较甲烷氧化菌丰度差异,分析环境因子对甲烷氧化菌分布的影响。【结果】土壤样品的理化性质测定发现江汉盆地样品的含水量最高,约22.8%,硝态氮平均含量最高位于玉北油田,达到31.96 μg/g干重土壤,春光油田出现最高的SO₄^2- (6 425.0 mg/g干重土壤)及Cl^- (1 617.0 mg/g干重土壤)浓度。qPCR分析发现pmoA基因丰度仅为土壤样本总细菌丰度的0.77%,表明甲烷氧化菌绝对数量较少。16S rRNA基因测序发现type I型甲基八叠球菌属(Methylosarcina)、甲基嗜热菌属(Methylocaldum)、甲基球菌属(Methylococcus)与type II型的甲基胞囊菌属(Methylocystis) 4种主要的甲烷氧化菌,甲烷氧化菌中的优势菌属的相对丰度极低,最高仅为0.124%。甲烷氧化菌功能基因pmoA测序发现type II型的甲基胞囊菌属、甲基弯曲菌属(Methylosinus)为主要优势pmoA基因型。环境因子与甲烷氧化菌丰度相关性分析表明甲烷氧化菌绝对丰度与铵态氮、pH、颗粒大小、硫酸根和氯离子显著相关,而环境因子与甲烷氧化菌的相对丰度的相关性分析仅发现与颗粒大小、总氮、总磷、金属离子(Al、Fe、K、Ca、Mg、Mn、Zn和Cu)显著相关。甲烷氧化菌pmoA功能基因组成判别相关分析(discriminant correlation analysis, DCA)表明含水量(土壤湿度)、pH值、<2 μm的土壤颗粒、总氮及钙离子对甲烷氧化菌pmoA功能基因组成影响具有显著性。【结论】本研究通过对来自7个典型油气藏区域的土壤样品的甲烷氧化菌的绝对丰度与相对丰度进行分析,发现基于不同的测量方法,甲烷氧化菌的丰度存在较大差异,16S rRNA基因测序无法较为全面地反映土壤样本中优势甲烷氧化菌的群落结构,不同地理位置的甲烷氧化菌指示群落存在差异,在全国尺度下油气区及背景点均无特有的甲烷氧化菌指示群落。对环境因子与甲烷氧化菌相关性分析发现,基于不同的甲烷氧化菌丰度指标,环境因子的显著影响具有较大差别。     

Molecular determinants for the chemical activation of the warmth-sensitive TRPV3 channel by the natural monoterpenoid carvacrol

Transient receptor potential vanilloid 3 (TRPV3), robustly expressed in the skin, is a nonselective calcium-permeable cation channel activated by warm temperature, voltage, and certain chemicals. Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel. However, how carvacrol activates TRPV3 mechanistically remains to be understood. Here, we describe the molecular determinants for chemical activation of TRPV3 by the agonist carvacrol. Patch clamp recordings reveal that carvacrol activates TRPV3 in a concentration-dependent manner, with an EC₅₀ of 0.2 mM, by increasing the probability of single-channel open conformation. Molecular docking of carvacrol into cryo-EM structure of TRPV3 combined with site-directed mutagenesis further identified a unique binding pocket formed by the channel S2-S3 linker important for mediating this interaction. Within the binding pocket consisting of four residues (Ile505, Leu508, Arg509, and Asp512), we report that Leu508 is the most critical residue for the activation of TRPV3 by carvacrol, but not 2-APB, a widely used nonspecific agonist and TRP channel modulator. Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3. We also propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3 modulators for the further understanding of TRPV3 channel pharmacology.     

DNA barcode reveals candidate species of Scinax and Ololygon (Anura: Hylidae) in Atlantic Forest

Molecular species delimitation methods are efficient tools to identify species, including the discovery of new taxa and cryptic organisms, thus being useful to biodiversity studies. In the present work, 16S mitochondrial sequences and cytochrome oxidase I (COI) were used to evaluate the richness of species in the genus Scinax and Ololygon from a biodiversity hotspot in Atlantic Forest. A total of 109 specimens formally belonging to eight species of Scinax and three species of Ololygon were collected in 13 localities along the state of Bahia (northeastern Brazil) and one site in Espírito Santo (southeastern Brazil). Of the Scinax species collected in this study, three were morphologically differentiated from other described species and identified as putative new species (Scinax sp.1, Scinax sp.2 and Scinax sp.3). The species delimitations were inferred using three different methods: ABGD, PTP and mPTP which allowed recognizing 11 Scinax species and five Ololygon species. Scinax sp. 1, Scinax sp. 2 and Scinax sp. 3, have been confirmed as new putative species and Ololygon argyreornata possibly contains cryptic species. We suggest additional studies, including morphological and bioacoustic data to validate these new putative species.     

Computer-Assisted Discovery of Alkaloids with Schistosomicidal Activity

Schistosomiasis is a chronic parasitic disease caused by trematodes of the genus Schistosoma; it is commonly caused by Schistosoma mansoni, which is transmitted by Bioamphalaria snails. Studies show that more than 200 million people are infected and that more than 90% of them live in Africa. Treatment with praziquantel has the best cost–benefit result on the market. However, hypersensitivity, allergy, and drug resistance are frequently presented after administration. From this perspective, ligand-based and structure-based virtual screening (VS) techniques were combined to select potentially active alkaloids against S. mansoni from an internal dataset (SistematX). A set of molecules with known activity against S. mansoni was selected from the ChEMBL database to create two different models with accuracy greater than 84%, enabling ligand-based VS of the alkaloid bank. Subsequently, structure-based VS was performed through molecular docking using four targets of the parasite. Finally, five consensus hits (i.e., five alkaloids with schistosomicidal potential), were selected. In addition, in silico evaluations of the metabolism, toxicity, and drug-like profile of these five selected alkaloids were carried out. Two of them, namely, 11,12-methylethylenedioxypropoxy and methyl-3-oxo-12-methoxy-n(1)-decarbomethoxy-14,15-didehydrochanofruticosinate, had plausible toxicity, metabolomics, and toxicity profiles. These two alkaloids could serve as starting points for the development of new schistosomicidal compounds based on natural products.