The Molecular Mechanism Underlying Mechanical Anisotropy of the Protein GB1

Mechanical responses of elastic proteins are crucial for their biological function and nanotechnological use. Loading direction has been identified as one key determinant for the mechanical responses of proteins. However, it is not clear how a change in pulling direction changes the mechanical unfolding mechanism of the protein. Here, we combine protein engineering, single-molecule force spectroscopy, and steered molecular dynamics simulations to systematically investigate the mechanical response of a small globular protein GB1. Force versus extension profiles from both experiments and simulations reveal marked mechanical anisotropy of GB1. Using native contact analysis, we relate the mechanically robust shearing geometry with concurrent rupture of native contacts. This clearly contrasts the sequential rupture observed in simulations for the mechanically labile peeling geometry. Moreover, we identify multiple distinct mechanical unfolding pathways in two loading directions. Implications of such diverse unfolding mechanisms are discussed. Our results may also provide some insights for designing elastomeric proteins with tailored mechanical properties.     

The Molecular Mechanism Underlying Recruitment and Insertion of Lipid-Anchored LC3 Protein into Membranes

Lipid modification of cytoplasmic proteins initiates membrane engagement that triggers diverse cellular processes. Despite the abundance of lipidated proteins in the human proteome, the key determinants underlying membrane recognition and insertion are poorly understood. Here, we define the course of spontaneous membrane insertion of LC3 protein modified with phosphatidylethanolamine using multiple coarse-grain simulations. The partitioning of the lipid anchor chains proceeds through a concerted process, with its two acyl chains inserting one after the other. Concurrently, a conformational rearrangement involving the α-helix III of LC3, especially in the three basic residues Lys⁶⁵, Arg⁶⁸, and Arg⁶⁹, ensures stable insertion of the phosphatidylethanolamine anchor into membranes. Mutational studies validate the crucial role of these residues, and further live-cell imaging analysis shows a substantial reduction in the formation of autophagic vesicles for the mutant proteins. Our study captures the process of water-favored LC3 protein recruitment to the membrane and thus opens, to our knowledge, new avenues to explore the cellular dynamics underlying vesicular trafficking.     

Self-Organization in Primates: Understanding the Rules Underlying Collective Movements

Patterns of collective movements, such as the distribution of leadership and the organization of individuals, may be either homogeneously (no leader, no specific order), or heterogeneously (1 or several leaders, and a highly stable order) distributed. Members of a group need to synchronize their activities and coordinate their movements, despite the fact that they differ in physiological or morphological traits. The degree of difference in these traits may affect their decision-making strategy. We demonstrate how a theoretical model based on a variation of a simple mimetic rule, i.e., an amplification process, can result in each of the various collective movement patterns and decision-making strategies observed in primates and other species. We consider cases in which 1) the needs of different individuals are identical and social relationships are equivalent between group members, 2) the needs of individuals are different and social relationships are equivalent, and 3) the needs of individuals are different and social relationships are different. Finally, 4) we assess how the synergy between 2 mimetism rules, specifically the probability of joining a movement and that of canceling an initiation, allows group members to stay synchronized and cohesive. Our models suggest that similar self-organized processes have been selected as reliable and well-adapted means for optimal collective decisions across species, despite differences in their biological and social characteristics.     

Active Cage Mechanism of Chaperonin-Assisted Protein Folding Demonstrated at Single-Molecule Level

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli have an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations (100 pM) to exclude aggregation, we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP) by single-pair fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We find that GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. Accelerated folding is achieved by encapsulation of folding intermediate in the GroEL/ES cage, independent of repetitive cycles of protein binding and release from GroEL. Moreover, photoinduced electron transfer experiments provided direct physical evidence that the confining environment of the chaperonin restricts polypeptide chain dynamics. This effect is mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. EL(KKK2)/ES functions as a passive cage in which folding occurs at the slow spontaneous rate. Taken together our findings suggest that protein encapsulation can accelerate folding by entropically destabilizing folding intermediates, in strong support of an active chaperonin mechanism in the folding of some proteins. Accelerated folding is biologically significant as it adjusts folding rates relative to the speed of protein synthesis.     

The Molecular Mechanism Underlying Morphine-Induced Akt Activation: Roles of Protein Phosphatases and Reactive Oxygen Species

Although Akt is reported to play a role in morphine’s cardioprotection, little is known about the mechanism underlying morphine-induced Akt activation. This study aimed to define the molecular mechanism underlying morphine-induced Akt activation and to determine if the mechanism contributes to the protective effect of morphine on ischemia/reperfusion injury. In cardiac H9c2 cells, morphine increased Akt phosphorylation at Ser⁴⁷³, indicating that morphine upregulates Akt activity. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling, was not involved in the action of morphine on Akt activity. Morphine decreased the activity of PP2A, a major protein Ser/Thr phosphatase, and inhibition of PP2A with okadaic acid (OA) mimicked the effect of morphine on Akt activity. The effects of morphine on PP2A and Akt activities were inhibited by the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)glycine (MPG) and the mitochondrial K_ATP channel closer 5-hydroxydecanoate (5HD). In support, morphine could produce ROS and this was reversed by 5HD. Finally, the cardioprotective effect of morphine on ischemia–reperfusion injury was mimicked by OA but was suppressed by 5HD or MPG, indicating that protein phosphatases and ROS are involved in morphine’s protection. In conclusion, morphine upregulates Akt activity by inactivating protein Ser/Thr phosphatases via ROS, which may contribute to the cardioprotective effect of morphine.     

Underlying Hydrophobic Sequence Periodicity of Protein Tertiary Structure

Abstract

Hydropathy plots or window averages over local stretches of the sequence of residue hydrophobicity have revealed patterns related to various protein tertiary structural features. This has enabled identification of regions of the sequence that are at the surface or within the interior of globular soluble proteins, regions located within the lipid bilayer of transmembrane proteins, portions of the sequence that characterize repeating motifs, as well as motifs that usefully characterize different protein structural families. This, therefore, provides one example of the generally expressed maxim that “sequence determines structure”. On the other hand, a number of previous investigations have shown the rapidly varying values of residue hydrophobicity along the sequence to be distributed approximately randomly. So one might question just how much of the sequence actually determines structure. It is, therefore, of interest to extract that part of this rapidly varying distribution of residue hydrophobicity that is responsible for the longer wavelength variations that correlate with protein tertiary structural features and to determine their prevalence within the entire distribution. This is accomplished by a finite Fourier analysis of the sequence of residue hydrophobicity and of a new measure of residue distance from the protein interior. Calculations are performed on a number of globins, immunoglobulins, cuprodoxins, and papain-like structures. The spectral power of the Fourier amplitudes of the frequencies extracted, whose inverse transforms underlie the windowed values of residue hydrophobicity is shown to be a small fraction of the total power of the hydrophobicity distribution and thereby consistent with a distribution that might appear to be predominantly random. The wide range of sequence identity between proteins having the same fold, all exhibiting similar small fractions of power amplitude that correlate with the longer wavelength inside-to- outside excursions of the amino acid residues, supports the general contention that close sequence identity is an expression of a close evolutionary relationship rather than an expression of structural similarity. Practical implications of the present analysis for protein structure prediction and engineering are also described.     

植物响应联合胁迫机制的研究进展

自然界中, 植物通常面对多重联合胁迫。在全球气候变化日益加剧的背景下, 多重联合胁迫对植物生长发育及作物产量形成的不利影响日益显著。阐明植物响应和适应联合胁迫的生理与分子机制, 对人们理解植物对自然环境的适应机理, 及培育耐受联合胁迫的新品种有重要意义。研究表明, 植物响应联合胁迫的机制是特异的, 不能简单地从单一胁迫响应叠加来推断。植物遭受联合胁迫时, 各种生理、代谢和信号途径相互作用, 使得植物响应联合胁迫非常复杂。该文综述了植物响应联合胁迫的生理与分子机理的最新进展, 并阐述了植物响应联合胁迫的研究方法。     

Structural Determinants Underlying Photoprotection in the Photoactive Orange Carotenoid Protein of Cyanobacteria

The photoprotective processes of photosynthetic organisms involve the dissipation of excess absorbed light energy as heat. Photoprotection in cyanobacteria is mechanistically distinct from that in plants; it involves the orange carotenoid protein (OCP), a water-soluble protein containing a single carotenoid. The OCP is a new member of the family of blue light-photoactive proteins; blue-green light triggers the OCP-mediated photoprotective response. Here we report structural and functional characterization of the wild type and two mutant forms of the OCP, from the model organism Synechocystis PCC6803. The structural analysis provides high resolution detail of the carotenoid-protein interactions that underlie the optical properties of the OCP, unique among carotenoid-proteins in binding a single pigment per polypeptide chain. Collectively, these data implicate several key amino acids in the function of the OCP and reveal that the photoconversion and photoprotective responses of the OCP to blue-green light can be decoupled.     

植物响应联合胁迫机制的研究进展

自然界中, 植物通常面对多重联合胁迫。在全球气候变化日益加剧的背景下, 多重联合胁迫对植物生长发育及作物产量形成的不利影响日益显著。阐明植物响应和适应联合胁迫的生理与分子机制, 对人们理解植物对自然环境的适应机理, 及培育耐受联合胁迫的新品种有重要意义。研究表明, 植物响应联合胁迫的机制是特异的, 不能简单地从单一胁迫响应叠加来推断。植物遭受联合胁迫时, 各种生理、代谢和信号途径相互作用, 使得植物响应联合胁迫非常复杂。该文综述了植物响应联合胁迫的生理与分子机理的最新进展, 并阐述了植物响应联合胁迫的研究方法。     

组学技术揭示水稻杂种优势遗传机制

杂种优势是杂交后代在生长或生殖性状上表现出优于亲本的现象。虽然杂种优势在农业生产上已广为应用,但其分子机理仍不清楚。最近,中国科学家通过分析17个代表性杂交稻(Oryza sativa)品种,共10 074个F2个体的全基因组序列和表型,对水稻产量杂种优势相关位点进行了系统定位和解析。此外,中国另一个科研小组通过整合杂交稻亲本和杂交种的表型组、转录组及基因组等多层次数据,深入研究了超级杂交稻两优培九产量的杂种优势基础。这些研究不仅为杂种优势理论的建立提供了新数据,也为水稻育种实践提供了有益的指导。     

有无反馈对欺骗过程的神经机制的调控作用

欺骗行为会导致欺骗结果的产生,欺骗结果又会直接影响欺骗行为的发生及其内在机制.虽然有研究表明,欺骗结果会对相应的欺骗过程产生调控作用,但对这一调控作用的机制并不清楚.本研究采用功能核磁共振技术,对两组被试分别使用有、无反馈(欺骗结果)的GKT范式并记录两组被试在进行诚实反应和欺骗反应时的大脑激活模式.结果发现,有反馈组与无反馈组相比,有反馈组的诚实反应和欺骗反应都导致了左侧顶叶皮层、左背部前扣带皮层、左侧脑岛、双侧视皮层和右侧小脑的更大激活;对两组而言,欺骗反应和诚实反应都导致了右腹外侧前额区域、双侧缘上回、左侧脑岛、右后内侧额叶、右侧颞中回和右侧纹状体的更大激活;此外,与无反馈组相比,有反馈组的欺骗反应与诚实反应在双侧纹状体和左侧脑岛上的激活差异更加明显.这些结果表明,有无欺骗结果对欺骗过程的神经机制具有调控作用,当需要面临欺骗结果时,欺骗过程将更大程度地涉及到奖赏预期和风险厌恶过程的参与.     

Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice

The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1^G576V). The GA-dependent degradation of SLR1^G576V was reduced in Slr1-d4, and compared with SLR1, SLR1^G576V showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1^G576V interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.     

rBTI及其突变体对C. elegans寿命的影响

重组荞麦胰蛋白酶抑制剂（recombinant buckwheat trypsin inhibitor,rBTI）是一种来源于荞麦Potato Ⅰ抑制剂家族的丝氨酸蛋白酶抑制剂,具有很好的生物活性及功能。先前的研究表明,rBTI在秀丽隐杆线虫（Caenorhabditis elegans）中具有很好的延长寿命的性质,但其具体的作用机制还不太清楚。本文的研究证明,rBTI能够调节转录因子DAF-16的转录活性,进而影响线虫的寿命,且该性质与其胰蛋白酶抑制活性密切相关。通过定点突变技术,分别对rBTI的45位、53位和44位氨基酸活性位点进行突变,获得了4种不同胰蛋白酶抑制活性的rBTI突变体,分别命名为rBTI-R45A,rBTI-R45F,rBTI-W53R和rBTI-P44T。经典模式生物秀丽隐杆线虫寿命检测实验显示,野生型rBTI可以明显延长C.elegans的寿命,且在0～10 μmol/L 范围内具有浓度依赖性。和未处理对照组相比,10 μmol/L 野生型rBTI延长寿命幅度可达到14.5%,但是突变体rBTI-R45A,rBTI-R45F和rBTI W53R均不同程度失去了延长寿命的功能。利用荧光显微观察及qRT-PCR等方法进一步研究发现,野生型rBTI 可增强寿命调控转录因子DAF-16的转录活性。与寿命检测实验结果一致,4种 rBTI突变体均不能使DAF-16转录活性增强。上述结果表明,在C.elegans中,rBTI可增强长寿因子DAF 16的转录活性,进而延长虫体寿命,且该功能的发挥依赖于其适当的胰蛋白酶抑制活性。本文的结果为进一步研究开发rBTI的功能提供了实验支持和理论基础。     

锌转运蛋白ZIP8 在骨关节炎中的表达及其机制研究

目的:探讨锌转运蛋白ZIP8在骨关节炎患者中的表达及其对软骨细胞生长及基质金属蛋白酶(MMPs)表达的影响。方法:收集20例骨关节炎患者(OA组)和20例非骨关节炎患者(对照组)血清和软骨组织;采用原子吸收分光光度计测定患者血清和软骨组织中锌离子的表达水平;MTT方法检测软骨细胞的生长活力;采用小RNA干扰沉默ZIP8基因的表达;实时荧光定量PCR方法检测ZIP8及金属基质蛋白酶MMP3、MMP9、MMP12和MMP13等基因的m RNA表达水平;蛋白免疫印迹检测ZIP8及MMP3、MMP9、MMP12和MMP13等蛋白的表达水平。结果:OA组的血清和软骨组织中的锌离子浓度明显高于对照组(P0.01)。OA组软骨组织中ZIP8的m RNA(P0.05)和蛋白(P0.01)表达水平显著高于对照组。ZIP8小RNA干扰片段可以有效的沉默ZIP的基因表达(P0.01);沉默ZIP8的表达促进骨关节炎患者来源的软骨细胞的生长(P0.05),并且降低基质金属蛋白酶包括MMP3,MMP9,MMP12和MMP13的表达水平(P0.05)。结论:ZIP8与骨关节炎密切相关,沉默ZIP8的表达可以提高软骨细胞的生长活力,并且抑制基质金属蛋白酶的表达,为骨关节炎的治疗提供了新的靶点。     

Underlying Mechanism of Quercetin-induced Cell Death in Human Glioma Cells

There has been considerable interest in recent years in the anti-tumor activities of flavonoids. Quercetin, a ubiquitous bioactive flavonoid, can inhibit proliferation and induce apoptosis in a variety of cancer cells. However, the precise molecular mechanism by which quercetin induces apoptosis in cancer cells is poorly understood. The present study was undertaken to examine the effect of quercetin on cell viability and to determine its underlying mechanism in human glioma cells. Quercetin resulted in loss of cell viability in a dose- and time-dependent manner and the decrease in cell viability was mainly attributed to cell death. Quercetin did not increase reactive oxygen species (ROS) generation and the quercetin-induced cell death was also not affected by antioxidants, suggesting that ROS generation is not involved in loss of cell viability. Western blot analysis showed that quercetin treatment caused rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Transient transfection with constitutively active forms of MEK and Akt protected against the quercetin-induced loss of cell viability. Quercetin-induced depolarization of mitochondrial membrane potential. Caspase activity was stimulated by quercetin and caspase inhibitors prevented the quercetin-induced loss of cell viability. Quercetin resulted in a decrease in expression of survivin, antiapoptotic proteins. Taken together, these findings suggest that quercetin results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of ERK, Akt, and survivin.     

Capturing the Mechanism Underlying TOP mRNA Binding to LARP1

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