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Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells (1,2), distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells (3). Given the complex choreography of the substrate through the secretion apparatus (4), the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex (1) connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems (1,4-6)., The canonical type IV secretion (T4S) system comprises 12 proteins, VirB1-11 and VirD4, and forms a large macromolecular complex that spans the cell envelope of Gram-negative bacteria (2). The hub [...]

The article discusses the serpinopathies have a diverse set of conformational diseases that involve the aberrant self-association of proteins to ordered aggregates. Topics include genetic variants such as the severe Z allele of 1-antitrypsin promote proteasomal degradation and the formation of ordered linear polymers; and the structure of the pathological polymers accumulate in patients has not been demonstrated.

Structural studies of bio-complexes using single particle cryo-Electron Microscopy (cryo-EM) is nowadays a well-established technique in structural biology and has become competitive with X-ray crystallography. Development of digital registration systems for electron microscopy images and algorithms for the fast and efficient processing of the recorded images and their following analysis has facilitated the determination of structures at near-atomic resolution. The latest advances in EM have enabled the determination of protein complex structures at 1.4-3 Å resolution for an extremely broad range of sizes (from ~100 kDa up to hundreds of MDa (Bartesaghi et al., Science 348(6239):1147-1151, 2015; Herzik et al., Nat Commun 10:1032, 2019; Wu et al., J Struct Biol X 4:100020, 2020; Zhang et al., Nat Commun 10:5511, 2019; Zhang et al., Cell Res 30(12):1136-1139, 2020; Yip et al., Nature 587(7832):157-161, 2020; https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year )). In 2022, nearly 1200 structures deposited to the EMDB database were at a resolution of better than 3 Å ( https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year ).To date, the highest resolutions have been achieved for apoferritin, which comprises a homo-oligomer of high point group symmetry (O432) and has rigid organization together with high stability (Zhang et al., Cell Res 30(12):1136-1139, 2020; Yip et al., Nature 587(7832):157-161, 2020). It has been used as a test object for the assessments of modern cryo-microscopes and processing methods during the last 5 years. In contrast to apoferritin bacterial secretion systems are typical examples of multi protein complexes exhibiting high flexibility owing to their functions relating to the transportation of small molecules, proteins, and DNA into the extracellular space or target cells. This makes their structural characterization extremely challenging (Barlow, Methods Mol Biol 532:397-411, 2009; Costa et al., Nat Rev Microbiol 13:343-359, 2015). The most feasible approach to reveal their spatial organization and functional modification is cryo-electron microscopy (EM). During the last decade, structural cryo-EM has become broadly used for the analysis of the bio-complexes that comprise multiple components and are not amenable to crystallization (Lyumkis, J Biol Chem 294:5181-5197, 2019; Orlova and Saibil, Methods Enzymol 482:321-341, 2010; Orlova and Saibil, Chem Rev 111(12):7710-7748, 2011).In this review, we will describe the basics of sample preparation for cryo-EM, the principles of digital data collection, and the logistics of image analysis focusing on the common steps required for reconstructions of both small and large biological complexes together with refinement of their structures to nearly atomic resolution. The workflow of processing will be illustrated by examples of EM analysis of Type IV Secretion System., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Thioredoxins (Trx) are ubiquitous multifunctional low-molecular weight proteins that together with thioredoxin reductases (TrxR) participate in the maintenance of protein thiol homeostasis in NADPH-dependent reactions. An increasing number of data reveal that the Trx-TrxR system is an attractive target for anticancer therapies. In this work, we have elaborated a new and simple synthetic approach employing Ugi reaction to synthesize several new inhibitors of this system. The influence of various electrophilic fragments of this new class of compounds on the inhibition of the Trx-TrxR system was evaluated. As a result, a new compound 19a (SK053), which inhibits the activity of the Trx-TrxR system and exhibits antitumor activity, was obtained. Biologic analyses revealed that 19a inhibits induction of NF-κB and AP-1 and decreases H(2)O(2) scavenging capacity in tumor cells. Altogether, we show that 19a is a novel potential antitumor peptidomimetic inhibitor that can be used as a starting compound for further optimization.