Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting.

Haematopoiesis dysregulation with the presence of immature myeloid and erythroid immunosuppressive cells are key characteristics of the immune escape phase of tumour development. Here, the role of in vitro generated B16F10 tumour cell-derived extracellular vesicles (tEVs) as indirect cellular communicators, participating in tumour-induced dysregulation of haematopoiesis, was explored. The isolated tEVs displayed features of small EVs with a size range of 100-200 nm, expressed the common EV markers CD63, CD9, and Alix, and had a spherical shape with a lipid bilayer membrane.

A metastasis-associated Pannexin1 mutant (Panx1 ) forms a minimalist ATP release channel.

A truncated form of the ATP release channel pannexin 1 (Panx1), Panx1 , is enriched in metastatic breast cancer cells and has been proposed to mediate metastatic cell survival by increasing ATP release through mechanosensitive Panx1 channels. However, whether Panx1 on its own (without the presence of wtPanx1) mediates ATP release has not been tested. Here, we show that Panx1 by itself can form a constitutively active membrane channel, capable of releasing ATP even in the absence of wild type Panx1.

Silk Assembly against Hydrophobic Surfaces─Modeling and Imaging of Formation of Nanofibrils.

A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces.

Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC).

Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1β release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell.

The long noncoding RNA ADIPINT regulates human adipocyte metabolism via pyruvate carboxylase.

The pleiotropic function of long noncoding RNAs is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase, a pivotal enzyme in energy metabolism. We developed an approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation, which identifies that ADIPINT binds to pyruvate carboxylase and validated the interaction with electron microscopy.

A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells and .

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo.

Optimizing purification of the peripheral membrane protein FAM92A1 fused to a modified spidroin tag.

Cryo-electron microscopy has revolutionized structural biology. In particular structures of proteins at the membrane interface have been a major contribution of cryoEM. Yet, visualization and characterization of peripheral membrane proteins remains challenging; mostly because there is no unified purification strategy for these proteins.

Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces.

Over the last few years, cryo electron microscopy has become the most important method in structural biology. While 80% of deposited maps are from single particle analysis, electron tomography has grown to become the second most important method. In particular sub-tomogram averaging has matured as a method, delivering structures between 2 and 5 Å from complexes in cells as well as in vitro complexes.

CryoEM reveals BIN1 (isoform 8) does not bind to single actin filaments .

Cells change their appearance by a concerted action of the cytoskeleton and the plasma membrane. The machinery that bends the membrane includes Bin/Amphiphysin/Rvs (BAR) domain proteins. Recently BAR domain proteins garnered attention as actin regulators, either by recruiting actin regulating proteins or through binding to actin directly.

Structure versus function: Are new conformations of pannexin 1 yet to be resolved?

Pannexin 1 (Panx1) plays a decisive role in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It is widely accepted that Panx1 exerts its role in the context of purinergic signaling by providing a transmembrane pathway for ATP. However, under certain conditions, Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability.

Structural basis for SARM1 inhibition and activation under energetic stress.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.

Cells Control BIN1-Mediated Membrane Tubulation by Altering the Membrane Charge.

The Bridging integrator 1 (BIN1)/Amphiphysin/Rvs (BAR) protein family is an essential part of the cell's machinery to bend membranes. BIN1 is a muscle-enriched BAR protein with an established role in muscle development and skeletal myopathies. Here, we demonstrate that BIN1, on its own, is able to form complex interconnected tubular systems in vitro, reminiscent of t-tubule system in muscle cells.

Structural Evidence for an Octameric Ring Arrangement of SARM1.

SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD+ to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy.

Regulated protein turnover: snapshots of the proteasome in action.

The ubiquitin proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and misfolded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques.

Protein-mediated transformation of lipid vesicles into tubular networks.

Key cellular processes are frequently accompanied by protein-facilitated shape changes in the plasma membrane. N-BAR-domain protein modules generate curvature by means of complex interactions with the membrane surface. The way they assemble and the mechanism by which they operate are largely dependent on their binding density.

Understanding the role of amphipathic helices in N-BAR domain driven membrane remodeling.

Endophilin N-BAR (N-terminal helix and Bin/amphiphysin/Rvs) domain tubulates and vesiculates lipid membranes in vitro via its crescent-shaped dimer and four amphipathic helices that penetrate into membranes as wedges. Like F-BAR domains, endophilin N-BAR also forms a scaffold on membrane tubes. Unlike F-BARs, endophilin N-BARs have N-terminal H0 amphipathic helices that are proposed to interact with other N-BARs in oligomer lattices.

Membrane curvature and its generation by BAR proteins.

Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the Bin/amphiphysin/Rvs (BAR) domain proteins.

Structural basis of membrane bending by the N-BAR protein endophilin.

Functioning as key players in cellular regulation of membrane curvature, BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices.

New insights into BAR domain-induced membrane remodeling.

Mesoscopic simulations and electron microscopy of N-BAR domain-induced liposome remodeling are used to characterize the process of liposome tubulation and vesiculation. The overall process of membrane remodeling is found to involve complex couplings among the N-BAR protein density, the degree of N-BAR oligomerization, and the membrane density. A comparison of complex remodeled liposome structures from mesoscopic simulations with those measured by electron microscopy experiments suggests that the process of membrane remodeling can be described via an appropriate mesoscopic free energy framework.

Two conformational changes are associated with glutamate translocation by the glutamate transporter EAAC1.

Glutamate is transported across membranes by means of a carrier mechanism that is thought to require conformational changes of the transport protein. In this work, we have determined the thermodynamic parameters of glutamate and the Na+ binding steps to their extracellular binding sites along with the activation parameters of rapid, glutamate-induced processes in the transport cycle by analyzing the temperature dependence of glutamate transport at steady state and pre-steady state. Our results suggest that glutamate binding to the transporter is driven by a negative reaction enthalpy (DeltaH0 = -33 kJ/mol), whereas the tighter binding of the non-transportable inhibitor TBOA is caused by an additional increase in entropy.

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism.

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.