Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines.

High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations.

Roles of zinc in cancers: From altered metabolism to therapeutic applications.

Zinc (Zn) is a crucial trace element involved in various cellular processes, including oxidative stress, apoptosis and immune response, contributing to cellular homeostasis. Dysregulation of Zn homeostasis occurs in certain cancers. This review discusses the role of Zn in cancer and its associated components, such as Zn-related proteins, their potential as biomarkers and the use of Zn-based strategies for tumor treatment.

ATP7B-Deficient Hepatocytes Reveal the Importance of Protein Misfolding Induced at Low Copper Concentration.

Copper is a transition metal essential for human life. Its homeostasis is regulated in the liver, which delivers copper to the whole body and excretes its excess outside the organism in the feces through the bile. These functions are regulated within hepatocytes, and the ATP7B copper transporter is central to making the switch between copper use and excretion.

Structures of Silver Fingers and a Pathway to Their Genotoxicity.

Recently, we demonstrated that Ag can directly replace Zn in zinc fingers (ZFs). The cooperative binding of Ag to ZFs leads to a thermodynamically irreversible formation of silver clusters destroying the native ZF structure. Thus, a reported loss of biological function of ZF proteins is a likely consequence of such replacement.

Development, formulation, and cellular mechanism of a lipophilic copper chelator for the treatment of Wilson's disease.

Copper homeostasis is finely regulated in human to avoid any detrimental impact of free intracellular copper ions. Upon copper accumulation, biliary excretion is triggered in liver thanks to trafficking of the ATP7B copper transporter to bile canaliculi. However, in Wilson's disease this protein is mutated leading to copper accumulation.

Correlative transmission electron microscopy and high-resolution hard X-ray fluorescence microscopy of cell sections to measure trace element concentrations at the organelle level.

Metals are essential for life and their concentration and distribution in organisms are tightly regulated. Indeed, in their free form, most transition metal ions are toxic. Therefore, an excess of physiologic metal ions or the uptake of non-physiologic metal ions can be highly detrimental to the organism.

Imaging inorganic nanomaterial fate down to the organelle level.

Nanotoxicology remains an important and emerging field since only recent years have seen the improvement of biological models and exposure setups toward real-life scenarios. The appropriate analysis of nanomaterial fate in these conditions also required methodological developments in imaging to become sensitive enough and element specific. In the last 2-4 years, impressive breakthroughs have been achieved using electron microscopy, nanoscale secondary ion mass spectrometry, X-ray fluorescence microscopy, or fluorescent sensors.

Iron Dysregulation in Human Cancer: Altered Metabolism, Biomarkers for Diagnosis, Prognosis, Monitoring and Rationale for Therapy.

Iron (Fe) is a trace element that plays essential roles in various biological processes such as DNA synthesis and repair, as well as cellular energy production and oxygen transport, and it is currently widely recognized that iron homeostasis is dysregulated in many cancers. Indeed, several iron homeostasis proteins may be responsible for malignant tumor initiation, proliferation, and for the metastatic spread of tumors. A large number of studies demonstrated the potential clinical value of utilizing these deregulated proteins as prognostic and/or predictive biomarkers of malignancy and/or response to anticancer treatments.

Lectin recognition and hepatocyte endocytosis of GalNAc-decorated nanostructured lipid carriers.

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc).

Safer-by-design biocides made of tri-thiol bridged silver nanoparticle assemblies.

Silver nanoparticles (AgNPs) are efficient biocides increasingly used in consumer products and medical devices. Their activity is due to their capacity to release bioavailable Ag(i) ions making them long-lasting biocides but AgNPs themselves are usually easily released from the product. Besides, AgNPs are highly sensitive to various chemical environments that triggers their transformation, decreasing their activity.

Formation of highly stable multinuclear AgS clusters in zinc fingers disrupts their structure and function.

Silver (Ag(i)) binding to consensus zinc fingers (ZFs) causes Zn(ii) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgS clusters. A compact eight-membered AgS structure formed by a CCCC ZF is the first cluster example reported for a single biological molecule. Ag(i)-induced conformational changes of ZFs can, as a consequence, affect transcriptional regulation and other cellular processes.

Canalicular domain structure and function in matrix-free hepatic spheroids.

Liver is pivotal in organism metabolism. This organ is receiving nutriments from the portal vein and then storing, metabolizing, distributing in the circulation or excreting excess and xenobiotics in bile. Liver architecture and hepatocyte polarization are crucial to achieve these functions.

Dual function of UPF3B in early and late translation termination.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance pathway that recognizes and degrades mRNAs with premature termination codons (PTCs). The mechanisms underlying translation termination are key to the understanding of RNA surveillance mechanisms such as NMD and crucial for the development of therapeutic strategies for NMD-related diseases. Here, we have used a fully reconstituted translation system to probe the NMD proteins for interaction with the termination apparatus.

Textural, Structural and Biological Evaluation of Hydroxyapatite Doped with Zinc at Low Concentrations.

The present work was focused on the synthesis and characterization of hydroxyapatite doped with low concentrations of zinc (Zn:HAp) (0.01 < < 0.05).

Impact of labile metal nanoparticles on cellular homeostasis. Current developments in imaging, synthesis and applications.

Background: The use of nanomaterials is constantly increasing in electronics, cosmetics, food additives, and is emerging in advanced biomedical applications such as theranostics, bio-imaging and therapeutics. However their safety raises concerns and requires appropriate methods to analyze their fate in vivo.

Scope Of Review: In this review, we describe the current knowledge about the toxicity of labile metal (ZnO, CuO and Ag) nanoparticles (NPs) both at the organism and cellular levels, and describe the pathways that are triggered to maintain cellular homeostasis.

A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion.

The conserved SecYEG protein-conducting channel and the accessory proteins SecDF-YajC and YidC constitute the bacterial holo-translocon (HTL), capable of protein-secretion and membrane-protein insertion. By employing an integrative approach combining small-angle neutron scattering (SANS), low-resolution electron microscopy and biophysical analyses we determined the arrangement of the proteins and lipids within the super-complex. The results guided the placement of X-ray structures of individual HTL components and allowed the proposal of a model of the functional translocon.

Cell-Free Synthesis of Macromolecular Complexes.

Cell-free protein synthesis based on E. coli cell extracts has been described for the first time more than 50 years ago. To date, cell-free synthesis is widely used for the preparation of toxic proteins, for studies of the translation process and its regulation as well as for the incorporation of artificial or labeled amino acids into a polypeptide chain.

XAS Investigation of Silver(I) Coordination in Copper(I) Biological Binding Sites.

Silver(I) is an unphysiological ion that, as the physiological copper(I) ion, shows high binding affinity for thiolate ligands; its toxicity has been proposed to be due to its capability to replace Cu(I) in the thiolate binding sites of proteins involved in copper homeostasis. Nevertheless, the nature of the Ag(I)-thiolate complexes formed within cells is poorly understood, and the details of Ag(I) coordination in such complexes in physiologically relevant conditions are mostly unknown. By making use of X-ray absorption spectroscopy (XAS), we characterized the Ag(I) binding sites in proteins related to copper homeostasis, such as the chaperone Atox1 and metallothioneins (MTs), as well as in bioinspired thiolate Cu(I) chelators mimicking these proteins, in solution and at physiological pH.

A network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain regulates UPF1 substrate recruitment and phosphorylation.

Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1-8-9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking.

Structural and functional analysis of the three MIF4G domains of nonsense-mediated decay factor UPF2.

Nonsense-mediated decay (NMD) is a eukaryotic quality control pathway, involving conserved proteins UPF1, UPF2 and UPF3b, which detects and degrades mRNAs with premature stop codons. Human UPF2 comprises three tandem MIF4G domains and a C-terminal UPF1 binding region. MIF4G-3 binds UPF3b, but the specific functions of MIF4G-1 and MIF4G-2 are unknown.

Directly from the source: endogenous preparations of molecular machines.

Purification from a source enriched in large macromolecular machines with basic cellular function is still the method of choice in many cases. Such complexes occur in sufficiently high copy numbers in the cell and can be isolated using classical protein purification protocols. Although advanced DNA recombinant technologies and sophisticated overexpression strategies are available, many complexes like the ribosome, RNA polymerase II and membrane protein complexes involved in photosynthesis or in oxidative phosphorylation can only be purified from a rich source.

Contamination from an affinity column: an encounter with a new villain in the world of membrane-protein crystallization.

Attempts to crystallize AtNTT1, a chloroplast ATP/ADP transporter from Arabidopsis thaliana, revealed an unexpected contaminant, Strep-Tactin, a variant of streptavidin that was used during purification of the protein. Although it was present in very small amounts, crystals of Strep-Tactin were reproducibly grown from the AtNTT1 solution. AtNTT1 was overexpressed in Escherichia coli and purified from detergent-solubilized membrane fractions using Strep-Tactin affinity chromatography based on an engineered streptavidin.

Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: toward a molecular understanding of the transport mechanism.

Background: Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters.

Methodology/principal Findings: In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana.

Expression of a chloroplast ATP/ADP transporter in E. coli membranes: behind the Mistic strategy.

Eukaryotic membrane protein expression is still a major bottleneck for structural studies. Production in E. coli often leads to low expression level and/or aggregated proteins.