Understanding the kinetic roles of the inducer heparin and of rod-like protofibrils during amyloid fibril formation by Tau protein

The aggregation of the natively disordered protein, Tau, to form lesions called neurofibrillary tangles is a characteristic feature of several neurodegenerative tauopathies. The polyanion, heparin, is commonly used as an inducer in studies of Tau aggregation in vitro, but there is surprisingly no comprehensive model describing, quantitatively, all aspects of the heparin-induced aggregation reaction. In this study, rate constants and extents of fibril formation by the four repeat domain of Tau (Tau4RD) have been reproducibly determined over a full range of heparin and protein concentrations. The kinetic role of heparin in the nucleation-dependent fibril formation reaction is shown to be limited to participation in the initial rate-limiting steps; a single heparin molecule binds two Tau4RD molecules, forming an aggregation-competent protein dimer, which then serves as a building block for further fibril growth. Importantly, the minimal kinetic model that is proposed can quantitatively account for the characteristic bell-shaped dependence of the aggregation kinetics on the stoichiometry of protein to heparin. Very importantly, this study also identifies for the first time short and thin, rod-like protofibrils that are populated transiently, early during the time course of fibril formation. The identification of these protofibrils as bona fide off-pathway species has implications for the development of therapies for tauopathies based on driving fibril formation as a means of protecting the cell from smaller, putatively toxic aggregates.     

Charge requirements for proton gradient-driven translocation of anthrax toxin

Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (ΔpH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ΔpH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ΔpH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LF(N)) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LF(N) to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LF(N) translocate independently of the ΔpH driving force. Acidic residues markedly increase the rate of ΔpH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ΔpH-driven unfolding and translocation. We discuss a ΔpH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the "molecular teeth" of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ΔpH translocation.     

Atomic force microscopy of Connexin40 gap junction hemichannels reveals calcium-dependent three-dimensional molecular topography and open-closed conformations of both the extracellular and cytoplasmic faces

Atomic force microscopy was used to study the three-dimensional molecular topography and calcium-sensitive conformational changes of Connexin40 hemichannels (connexons) reconstituted in 1,2-dioeloyl-sn-glycero-3-phosphatidylcholine lipid bilayers. Two classes of objects were observed that differed in their protrusion heights above the bilayer (2.6 versus 4.2 nm). Comparison to reconstituted connexons containing Connexin40 truncated to eliminate most of its C-terminal cytoplasmic domain showed that the two height classes corresponded to the shorter extracellular and taller cytoplasmic aspects of the hemichannels and that the C-terminal tail of Connexin40 contributes ～1.6 nm in thickness. Hemichannels imaged in solutions containing < 10 μm Ca(2+) showed 3.1-3.2 nm depressions (openings) in 30% of the cytoplasmic faces and 65% of the extracellular faces, and high-resolution three-dimensional topography of extracellular or cytoplasmic aspects of some connexons was observed. After addition of 3.6 mm Ca(2+), > 75% of the connexons in either orientation adopted closed conformations. In contrast, hemichannels imaged in the presence of 0.1 mm EDTA showed large (5.6- to 5.8-nm diameter) openings in nearly all hemichannels regardless of orientation, and detailed topography was visible in many connexons. Real-time imaging following the addition of 3.6 mm Ca(2+) showed transitions of both extracellular and cytoplasmic orientations from "open" into "closed" conformations within several minutes. These studies provide the first high-resolution topographic information regarding a connexin with a large cytoplasmic domain and suggest that the extramembranous portions of Connexin40 contribute to a channel entrance that is relaxed by chelation of residual divalent cations.     

L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotide-binding oligomerization domain 1

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 μM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 μM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 μM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.     

Spectrin-like repeats 11-15 of human dystrophin show adaptations to a lipidic environment

Dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. In the present work, we characterized the behavior of dystrophin 11-15 (DYS R11-15), five spectrin-like repeats from the central domain of human dystrophin, with lipids. DYS R11-15 displays an amphiphilic character at the liquid/air interface while maintaining its secondary α-helical structure. The interaction of DYS R11-15 with small unilamellar vesicles (SUVs) depends on the lipid nature, which is not the case with large unilamellar vesicles (LUVs). In addition, switching from anionic SUVs to anionic LUVs suggests the lipid packing as a crucial factor for the interaction of protein and lipid. The monolayer model and the modulation of surface pressure aim to mimic the muscle at work (i.e. dynamic changes of muscle membrane during contraction and relaxation) (high and low surface pressure). Strikingly, the lateral pressure modifies the protein organization. Increasing the lateral pressure leads the proteins to be organized in a regular network. Nevertheless, a different protein conformation after its binding to monolayer is revealed by trypsin proteolysis. Label-free quantification by nano-LC/MS/MS allowed identification of the helices in repeats 12 and 13 involved in the interaction with anionic SUVs. These results, combined with our previous studies, indicate that DYS R11-15 constitutes the only part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid packing and lipid nature. We provide strong experimental evidence for a physiological role of the central domain of dystrophin in sarcolemma scaffolding through modulation of lipid-protein interactions.     

Structural investigations on native collagen type I fibrils using AFM

This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.     

Role of a conserved salt bridge between the PAS core and the N-terminal domain in the activation of the photoreceptor photoactive yellow protein

The effect of ionic strength on the conformational equilibrium between the I(2) intermediate and the signaling state I(2)' of the photoreceptor PYP and on the rate of recovery to the dark state were investigated by time-resolved absorption and fluorescence spectroscopy. With increasing salt concentration up to approximately 600 mM, the recovery rate k(3) decreases and the I(2)/I(2)' equilibrium (K) shifts in the direction of I(2)'. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl(2), MgSO(4)) salts show that the low salt effect depends on the ionic strength and not on the cation or anion species. These observations can be described over the entire ionic strength range by considering the activity coefficients of an interdomain salt bridge. At low ionic strength the activity coefficient decreases due to counterion screening whereas at high ionic strength binding of water by the salt leads to an increase in the activity coefficient. From the initial slopes of the plots of log k(3) and log K versus the square root of the ionic strength, the product of the charges of the interacting groups was found to be -1.3 +/- 0.2, suggesting a monovalent ion pair. The conserved salt bridge K110/E12 connecting the beta-sheet of the PAS core and the N-terminal domain is a prime candidate for this ion pair. To test this hypothesis, the mutants K110A and E12A were prepared. In K110A the salt dependence of the I(2)/I(2)' equilibrium was eliminated and of the recovery rate was greatly reduced below approximately 600 mM. Moreover, at low salt the recovery rate was six times slower than in wild-type. In E12A significant salt dependence remained, which is attributed to the formation of a novel salt bridge between K110 and E9. At high salt reversal occurs in both mutants suggesting that salting out stabilizes the more compact I(2) structure. However, chaotropic anions like SCN shift the I(2)/I(2)' equilibrium toward the partially unfolded I(2)' form. The salt linkage K110/E12 stabilizes the photoreceptor in the inactive state in the dark and is broken in the light-induced formation of the signaling state, allowing the N-terminal domain to detach from the beta-scaffold PAS core.     

Meal sharing among the Ye’kwana

In this study meal sharing is used as a way of quantifying food transfers between households. Traditional food-sharing studies measure the flow of resources between households. Meal sharing, in contrast, measures food consumption acts according to whether one is a host or a guest in the household as well as the movement of people between households in the context of food consumption. Our goal is to test a number of evolutionary models of food transfers, but first we argue that before one tests models of who should receive food one must understand the adaptiveness of food transfers. For the Ye’kwana, economies of scale in food processing and preparation appear to set the stage for the utility of meal sharing. Evolutionary models of meal sharing, such as kin selection and reciprocal altruism, are evaluated along with non-evolutionary models, such as egalitarian exchange and residential propinquity. In addition, a modified measure of exchange balance—proportional balance—is developed. Reciprocal altruism is shown to be the strongest predictor of exchange intensity and balance.     

中国蜥蜴类生活史和生态学特征数据集

截至2020年底, 中国共有226种蜥蜴类(不包括外来入侵种), 是世界上蜥蜴类多样性最丰富的国家之一。系统整理中国现有蜥蜴类的特征数据在物种起源与进化、形成与灭绝、保护生物学等研究中具有重要意义。但是, 目前还没有关于我国蜥蜴类生活史、生态学和地理分布等物种特征的完整数据库。本文通过系统查阅文献和数据资料, 共收集整理了中国现有226种本土蜥蜴类19个特征数据: 描述年份、中国受威胁等级、全球受威胁等级、是否中国特有种、是否岛屿特有种、平均体长、平均体重、食性、窝卵数、繁殖模式、四肢发育、活动时间、栖息生境、栖息地类型、栖息地宽度、海拔分布范围、地理分布范围、动物地理界和分布省份。在上述特征中, 除了四肢发育、描述年份、是否中国特有种、是否岛屿特有种和分布省份外, 其余特征数据均存在不同程度的缺失, 数据完整度为47.14%-100%。本数据集是目前关于中国蜥蜴类最新和最全的物种特征数据库, 可为我国蜥蜴类生态学、进化生物学、生物地理学和保护生物学等研究领域提供数据支持。     

Capturing actin assemblies in cells using in situ cryo-electron tomography

Actin contributes to an exceptionally wide range of cellular processes through the assembly and disassembly of highly dynamic and ordered structures. Visualizing these structures in cells can help us understand how the molecular players of the actin machinery work together to produce force-generating systems. In recent years, cryo-electron tomography (cryo-ET) has become the method of choice for structural analysis of the cell interior at the molecular scale. Here we review advances in cryo-ET workflows that have enabled this transformation, especially the automation of sample preparation procedures, data collection, and processing. We discuss new structural analyses of dynamic actin assemblies in cryo-preserved cells, which have provided mechanistic insights into actin assembly and function at the nanoscale. Finally, we highlight the latest visual proteomics studies of actin filaments and their interactors reaching sub-nanometer resolutions in cells.