Post-Translational Modifications Control Phase Transitions of Tau.

The self-assembly of Tau(297-391) into filaments, which mirror the structures observed in Alzheimer's disease (AD) brains, raises questions about the role of AD-specific post-translational modifications (PTMs) in the formation of paired helical filaments (PHFs). To investigate this, we developed a synthetic approach to produce Tau(291-391) featuring N-acetyllysine, phosphoserine, phosphotyrosine, and N-glycosylation at positions commonly modified in post-mortem AD brains, thus facilitating the study of their roles in Tau pathology. Using transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and a range of optical microscopy techniques, we discovered that these modifications generally hinder the in vitro assembly of Tau into PHFs. Interestingly, while acetylation's effect on Tau assembly displayed variability, either promoting or inhibiting phase transitions in the context of cofactor free aggregation, heparin-induced aggregation, and RNA-mediated liquid-liquid phase separation (LLPS), phosphorylation uniformly mitigated these processes. Our observations suggest that PTMs, particularly those situated outside the fibril's rigid core are pivotal in the nucleation of PHFs. Moreover, in scenarios involving heparin-induced aggregation leading to the formation of heterogeneous aggregates, most AD-specific PTMs, except for K311, appeared to decelerate the aggregation process. The impact of acetylation on RNA-induced LLPS was notably site-dependent, exhibiting both facilitative and inhibitory effects, whereas phosphorylation consistently reduced LLPS across all proteoforms examined. These insights underscore the complex interplay between site-specific PTMs and environmental factors in modulating Tau aggregation kinetics, enhancing our understanding of the molecular underpinnings of Tau pathology in AD and highlighting the critical role of PTMs located outside the ordered filament core in driving the self-assembly of Tau into PHF structures.

A simple flash and freeze system for cryogenic time-resolved electron microscopy.

As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies.

Progress and gaps of extracellular vesicle-mediated intercellular cargo transfer in the central nervous system.

A fundamentally novel function proposed for extracellular vesicles (EVs) is to transfer bioactive molecules in intercellular signaling. In this minireview, we discuss recent progress on EV-mediated cargo transfer in the central nervous system (CNS) and major gaps in previous studies. We also suggest a set of experiments necessary for bridging the gaps and establishing the physiological roles of EV-mediated cargo transfer.

Activation of the insulin receptor by an insulin mimetic peptide.

Insulin receptor (IR) signaling defects cause a variety of metabolic diseases including diabetes. Moreover, inherited mutations of the IR cause severe insulin resistance, leading to early morbidity and mortality with limited therapeutic options. A previously reported selective IR agonist without sequence homology to insulin, S597, activates IR and mimics insulin's action on glycemic control. To elucidate the mechanism of IR activation by S597, we determine cryo-EM structures of the mouse IR/S597 complex. Unlike the compact T-shaped active IR resulting from the binding of four insulins to two distinct sites, two S597 molecules induce and stabilize an extended T-shaped IR through the simultaneous binding to both the L1 domain of one protomer and the FnIII-1 domain of another. Importantly, S597 fully activates IR mutants that disrupt insulin binding or destabilize the insulin-induced compact T-shape, thus eliciting insulin-like signaling. S597 also selectively activates IR signaling among different tissues and triggers IR endocytosis in the liver. Overall, our structural and functional studies guide future efforts to develop insulin mimetics targeting insulin resistance caused by defects in insulin binding and stabilization of insulin-activated state of IR, demonstrating the potential of structure-based drug design for insulin-resistant diseases.

Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse.

Electron tomography of the chemical synapse provides important architectural information regarding the organization of synaptic organelles including synaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and biopsy punches to collect specific brain regions of interest suitable for subsequent preservation and EM tomographic imaging.

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines.

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.

Synergistic activation of the insulin receptor via two distinct sites.

Insulin receptor (IR) signaling controls multiple facets of animal physiology. Maximally four insulins bind to IR at two distinct sites, termed site-1 and site-2. However, the precise functional roles of each binding event during IR activation remain unresolved. Here, we showed that IR incompletely saturated with insulin predominantly forms an asymmetric conformation and exhibits partial activation. IR with one insulin bound adopts a Γ-shaped conformation. IR with two insulins bound assumes a Ƭ-shaped conformation. One insulin binds at site-1 and another simultaneously contacts both site-1 and site-2 in the Ƭ-shaped IR dimer. We further show that concurrent binding of four insulins to sites-1 and -2 prevents the formation of asymmetric IR and promotes the T-shaped symmetric, fully active state. Collectively, our results demonstrate how the synergistic binding of multiple insulins promotes optimal IR activation.

Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases.

Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.

Structural mechanism of muscle nicotinic receptor desensitization and block by curare.

Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.

Room for Two: The Synaptophysin/Synaptobrevin Complex.

Synaptic vesicle release is regulated by upwards of 30 proteins at the fusion complex alone, but disruptions in any one of these components can have devastating consequences for neuronal communication. Aberrant molecular responses to calcium signaling at the pre-synaptic terminal dramatically affect vesicle trafficking, docking, fusion, and release. At the organismal level, this is reflected in disorders such as epilepsy, depression, and neurodegeneration. Among the myriad pre-synaptic proteins, perhaps the most functionally mysterious is synaptophysin (SYP). On its own, this vesicular transmembrane protein has been proposed to function as a calcium sensor, a cholesterol-binding protein, and to form ion channels across the phospholipid bilayer. The downstream effects of these functions are largely unknown. The physiological relevance of SYP is readily apparent in its interaction with synaptobrevin (VAMP2), an integral element of the neuronal SNARE complex. SNAREs, soluble NSF attachment protein receptors, comprise a family of proteins essential for vesicle fusion. The complex formed by SYP and VAMP2 is thought to be involved in both trafficking to the pre-synaptic membrane as well as regulation of SNARE complex formation. Recent structural observations specifically implicate the SYP/VAMP2 complex in anchoring the SNARE assembly at the pre-synaptic membrane prior to vesicle fusion. Thus, the SYP/VAMP2 complex appears vital to the form and function of neuronal exocytotic machinery.

Purification of a native nicotinic receptor.

Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of pentameric ligand-gated ion channels. The electric organ of the Torpedo ray is extraordinarily rich in an acetylcholine receptor that is homologous to the human nicotinic receptor found at the neuromuscular junction. Due to this abundant natural source in the fish and the relatively accessible preparation of the neuromuscular junction (compared to a central synapse), this muscle-type receptor and specifically the fish receptors have long been used as the prototype for study of nicotinic receptors. However, an absence of structural detail at high resolution has limited the chemical interpretation of this archetypal nicotinic receptor. One of the main concerns in preparing receptor for high resolution structural analysis was its documented sensitivity to particular detergents and requirements for specific lipids in order to maintain function after reconstitution in a membrane. Here, we present methods for purifying native nicotinic receptor from Torpedo electric tissue that maintains functionality after reconstitution and that is amenable to high resolution structural analysis. The specific developments we describe include detergent exchange during purification, inclusion of specific lipids during purification and for nanodisc reconstitution, and synthesis of a new affinity reagent for rapid isolation of receptors.

SNARE Zippering Is Suppressed by a Conformational Constraint that Is Removed by v-SNARE Splitting.

Intracellular vesicle fusion is catalyzed by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Vesicle-anchored v-SNAREs pair with target membrane-associated t-SNAREs to form trans-SNARE complexes, releasing free energy to drive membrane fusion. However, trans-SNARE complexes are unable to assemble efficiently unless activated by Sec1/Munc18 (SM) proteins. Here, we demonstrate that SNAREs become fully active when the v-SNARE is split into two fragments, eliminating the requirement of SM protein activation. Mechanistically, v-SNARE splitting accelerates the zippering of trans-SNARE complexes, mimicking the stimulatory function of SM proteins. Thus, SNAREs possess the full potential to drive efficient membrane fusion but are suppressed by a conformational constraint. This constraint is removed by SM protein activation or v-SNARE splitting. We suggest that ancestral SNAREs originally evolved to be fully active in the absence of SM proteins. Later, a conformational constraint coevolved with SM proteins to achieve the vesicle fusion specificity demanded by complex endomembrane systems.

Pulse-Chase Proteomics of the App Knockin Mouse Models of Alzheimer's Disease Reveals that Synaptic Dysfunction Originates in Presynaptic Terminals.

Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer's disease (AD). To observe protein turnover at early stages of amyloid beta (Aß) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aß accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV)-associated proteins, have elevated levels, misfold in both a plaque-dependent and -independent manner, and interact with APP and Aß. Concurrent with elevated levels of SV-associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper's transparent peer review process is included in the Supplemental Information.

On-Chip Acousto Thermal Shift Assay for Rapid and Sensitive Assessment of Protein Thermodynamic Stability.

Thermal shift assays (TSAs) have been extensively used to study thermodynamics of proteins and provide an efficient means to assess protein-ligand binding or protein-protein interactions. However, existing TSAs have limitations, such as being time consuming, labor intensive, or having low sensitivity. Herein, an acousto thermal shift assay (ATSA), the first ultrasound enabled TSA, is reported for real-time analysis of protein thermodynamic stability. It capitalizes the coupling of unique acoustic mechanisms to achieve protein unfolding, concentration, and measurement on a single microfluidic chip within minutes. Compared to conventional TSA methods, the ATSA technique enables ultrafast (at least 30 times faster), highly sensitive (7-34 folds higher), and label-free monitoring of protein-ligand interactions and protein stability. ATSA paves new avenues for protein analysis in biology, medicine, and fast diagnosis.

Structure of the Native Muscle-type Nicotinic Receptor and Inhibition by Snake Venom Toxins.

The nicotinic acetylcholine receptor, a pentameric ligand-gated ion channel, converts the free energy of binding of the neurotransmitter acetylcholine into opening of its central pore. Here we present the first high-resolution structure of the receptor type found in muscle-endplate membrane and in the muscle-derived electric tissues of fish. The native receptor was purified from Torpedo electric tissue and functionally reconstituted in lipids optimal for cryo-electron microscopy. The receptor was stabilized in a closed state by the binding of α-bungarotoxin. The structure reveals the binding of a toxin molecule at each of two subunit interfaces in a manner that would block the binding of acetylcholine. It also reveals a closed gate in the ion-conducting pore, formed by hydrophobic amino acid side chains, located ∼60 Å from the toxin binding sites. The structure provides a framework for understanding gating in ligand-gated channels and how mutations in the acetylcholine receptor cause congenital myasthenic syndromes.

An isothermal shift assay for proteome scale drug-target identification.

Most small molecule drugs act on living systems by physically interacting with specific proteins and modulating target function. Identification of drug binding targets, within the complex milieu of the human proteome, remains a challenging task of paramount importance in drug discovery. Existing approaches for target identification employ complex workflows with limited throughput. Here, we present the isothermal shift assay (iTSA), a mass spectrometry method for proteome-wide identification of drug targets within lysates or living cells. Compared with prevailing methods, iTSA uses a simplified experimental design with increased statistical power to detect thermal stability shifts that are induced by small molecule binding. Using a pan-kinase inhibitor, staurosporine, we demonstrate improved performance over commonly used thermal proteome profiling methods, identifying known targets in cell lysates and living cells. We also demonstrate the identification of both known targets and additional candidate targets for the kinase inhibitor harmine in cell and tissue lysates.

Flattening of Diluted Species Profile via Passive Geometry in a Microfluidic Device.

In recent years, microfluidic devices have become an important tool for use in lab-on-a-chip processes, including drug screening and delivery, bio-chemical reactions, sample preparation and analysis, chemotaxis, and separations. In many such processes, a flat cross-sectional concentration profile with uniform flow velocity across the channel is desired to achieve controlled and precise solute transport. This is often accommodated by the use of electroosmotic flow, however, it is not an ideal for many applications, particularly biomicrofluidics. Meanwhile, pressure-driven systems generally exhibit a parabolic cross-sectional concentration profile through a channel. We draw inspiration from finite element fluid dynamics simulations to design and fabricate a practical solution to achieving a flat solute concentration profile in a two-dimensional (2D) microfluidic channel. The channel possesses geometric features to passively flatten the solute profile before entering the defined region of interest in the microfluidic channel. An obviously flat solute profile across the channel is demonstrated in both simulation and experiment. This technology readily lends itself to many microfluidic applications which require controlled solute transport in pressure driven systems.

Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE.

Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.

Synaptotagmin 17 controls neurite outgrowth and synaptic physiology via distinct cellular pathways.

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors.

Cryo-EM structure of OSCA1.2 from Oryza sativa elucidates the mechanical basis of potential membrane hyperosmolality gating.

Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.