Compounds derived from Humulus lupulus inhibit SARS-CoV-2 papain-like protease and virus replication.

BACKGROUND: Selected natural compounds exhibit very good antiviral properties. Especially, the medicinal plant Humulus lupulus (hop) contains several secondary plant metabolites some of which have previously shown antiviral activities. Among them, the prenylated chalcone xanthohumol (XN) demonstrated to be a potent inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). HYPOTHESIS/PURPOSE: Following the finding that xanthohumol (XN) is a potent inhibitor of SARS-CoV-2 Mpro, the effect of XN and its major derivatives isoxanthohumol (IXN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) from hops on SARS-CoV-2 papain-like protease (PLpro) were investigated. STUDY DESIGN: The modulatory effect of the hop compounds on PLpro were studied first in silico and then in vitro. In addition, the actual effect of hop compounds on the replication of SARS-CoV-2 in host cells was investigated. METHODS: In silico docking analysis was used to predict the binding affinity of hop compounds to the active site of PLpro. A recombinant PLpro was cloned, purified, characterized, and analyzed by small-angle X-ray scattering (SAXS), deISGylation assays, and kinetic analyses. Antiviral activity of hop compounds was assessed using the fluorescently labeled wildtype SARS-CoV-2 (icSARS-CoV-2-mNG) in Caco-2 host cells. RESULTS: Our in silico docking suggests that the purified hop compounds bind to the active site of SARS-CoV-2 PLpro blocking the access of its natural substrates. The hop-derived compounds inhibit SARS-CoV-2 PLpro with half maximal inhibitory concentration (IC50) values in the range of 59-162 µM. Furthermore, we demonstrate that XN and 6-PN, in particular, impede viral replication with IC50 values of 3.3 µM and 7.3 µM, respectively. CONCLUSION: In addition to the already known inhibition of Mpro by XN, our results show, for the first time, that hop-derived compounds target also SARS-CoV-2 PLpro which is a promising therapeutic target as it contributes to both viral replication and modulation of the immune system. These findings support the possibility to develop new hop-derived antiviral drugs targeting human coronaviruses.

USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

Functionality of the Na+-translocating NADH:quinone oxidoreductase and quinol:fumarate reductase from Prevotella bryantii inferred from homology modeling.

Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiota. In Prevotella bryantii, the Na+-translocating NADH:quinone oxidoreductase (NQR) and quinol:fumarate reductase (QFR) interact using menaquinone as electron carrier, catalyzing NADH:fumarate oxidoreduction. P. bryantii NQR establishes a sodium-motive force, whereas P. bryantii QFR does not contribute to membrane energization. To elucidate the possible mode of function, we present 3D structural models of NQR and QFR from P. bryantii to predict cofactor-binding sites, electron transfer routes and interaction with substrates. Molecular docking reveals the proposed mode of menaquinone binding to the quinone site of subunit NqrB of P. bryantii NQR. A comparison of the 3D model of P. bryantii QFR with experimentally determined structures suggests alternative pathways for transmembrane proton transport in this type of QFR. Our findings are relevant for NADH-dependent succinate formation in anaerobic bacteria which operate both NQR and QFR.

The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates.

Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.

Fast fragment and compound screening pipeline at the Swiss Light Source.

Crystallography-based fragment screening is a highly effective technique employed in structure-based drug discovery to expand the range of lead development opportunities. It allows screening and sorting of weakly binding, low molecular mass fragments, which can be developed into larger high-affinity lead compounds. Technical improvements at synchrotron beamlines, design of innovative libraries mapping chemical space efficiently, effective soaking methods and enhanced data analysis have enabled the implementation of high-throughput fragment screening pipelines at multiple synchrotron facilities. This widened access to CBFS beyond the pharma industry has allowed academic users to rapidly screen large quantities of fragment-soaked protein crystals. The positive outcome of a CBFS campaign is a set of structures that present the three-dimensional arrangement of fragment-protein complexes in detail, thereby providing information on the location and the mode of interaction of bound fragments. Through this review, we provide users with a comprehensive guide that sets clear expectations before embarking on a crystallography-based fragment screening campaign. We present a list of essential pre-requirements that must be assessed, including the suitability of your current crystal system for a fragment screening campaign. Furthermore, we extensively discuss the available methodological options, addressing their limitations and providing strategies to overcome them. Additionally, we provide a brief perspective on how to proceed once hits are obtained. Notably, we emphasize the solutions we have implemented for instrumentation and software development within our Fast Fragment and Compound Screening pipeline. We also highlight third-party software options that can be utilized for rapid refinement and hit assessment.

Conformational coupling of redox-driven Na+-translocation in Vibrio cholerae NADH:quinone oxidoreductase.

In the respiratory chain, NADH oxidation is coupled to ion translocation across the membrane to build up an electrochemical gradient. In the human pathogen Vibrio cholerae, the sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) generates a sodium gradient by a so far unknown mechanism. Here we show that ion pumping in Na+-NQR is driven by large conformational changes coupling electron transfer to ion translocation. We have determined a series of cryo-EM and X-ray structures of the Na+-NQR that represent snapshots of the catalytic cycle. The six subunits NqrA, B, C, D, E, and F of Na+-NQR harbor a unique set of cofactors that shuttle the electrons from NADH twice across the membrane to quinone. The redox state of a unique intramembranous [2Fe-2S] cluster orchestrates the movements of subunit NqrC, which acts as an electron transfer switch. We propose that this switching movement controls the release of Na+ from a binding site localized in subunit NqrB.

Concerning the photophysics of fluorophores towards tailored bioimaging compounds: a case study involving S100A9 inflammation markers.

A full understanding concerning the photophysical properties of a fluorescent label is crucial for a reliable and predictable performance in biolabelling applications. This holds true not only for the choice of a fluorophore in general, but also for the correct interpretation of data, considering the complexity of biological environments. In the frame of a case study involving inflammation imaging, we report the photophysical characterization of four fluorescent S100A9-targeting compounds in terms of UV-vis absorption and photoluminescence spectroscopy, fluorescence quantum yields (ΦF) and excited state lifetimes (τ) as well as the evaluation of the radiative and non-radiative rate constants (kr and knr, respectively). The probes were synthesized based on a 2-amino benzimidazole-based lead structure in combination with commercially available dyes, covering a broad color range from green (6-FAM) over orange (BODIPY-TMR) to red (BODIPY-TR) and near-infrared (Cy5.5) emission. The effect of conjugation with the targeting structure was addressed by comparison of the probes with their corresponding dye-azide precursors. Additionally, the 6-FAM and Cy5.5 probes were measured in the presence of murine S100A9 to determine whether protein binding influences their photophysical properties. An interesting rise in ΦF upon binding of 6-FAM-SST177 to murine S100A9 enabled the determination of its dissociation equilibrium constant, reaching up to KD = 324 nM. This result gives an outlook for potential applications of our compounds in S100A9 inflammation imaging and fluorescence assay developments. With respect to the other dyes, this study demonstrates how diverse microenvironmental factors can severely impair their performance while rendering them poor performers in biological media, showing that a preliminary photophysical screening is key to assess the suitability of a particular luminophore.

Fast IMAC purification of non-tagged S100A8/A9 (calprotectin) from Homo sapiens and Sus scrofa.

S100A8/A9 (calprotectin) is a damage-associated molecular pattern molecule (DAMP) that plays a key role in the innate immune response of mammalia. S100A8/A9 is therefore widely used as a biomarker in human and veterinary medicine, but diagnostic tools for the detection of S100A8/A9 are rarely optimised for the specific organism, since the corresponding S100A8/A9 is often not available. There is need for an easy, reliable protocol for the production of recombinant, highly pure S100A8/A9 from various mammalia. Here we describe the expression and purification of recombinant human and porcine S100A8/A9 by immobilized metal affinity chromatography (IMAC), which takes advantage of the intrinsic, high-affinity binding of native un-tagged S100A8/A9 to metal ions. Highly pure S100A8/A9 is obtained by a combination of IMAC, ion exchange and size exclusion chromatographic steps. Considering the high sequence homology and conservation of the metal ion coordinating residues of S100A8/A9 metal binding sites, the protocol is presumably applicable to S100A8/A9 of various mammalia.

Assessment of the Genetic Spectrum of Uncombable Hair Syndrome in a Cohort of 107 Individuals.

Importance: Uncombable hair syndrome (UHS) is a rare hair shaft anomaly that manifests during infancy and is characterized by dry, frizzy, and wiry hair that cannot be combed flat. Only about 100 known cases have been reported so far. Objective: To elucidate the genetic spectrum of UHS. Design, Setting, and Participants: This cohort study includes 107 unrelated index patients with a suspected diagnosis of UHS and family members who were recruited worldwide from January 2013 to December 2021. Participants of all ages, races, and ethnicities were recruited at referral centers or were enrolled on their own initiative following personal contact with the authors. Genetic analyses were conducted in Germany from January 2014 to December 2021. Main Outcomes and Measures: Clinical photographs, Sanger or whole-exome sequencing and array-based genotyping of DNA extracted from blood or saliva samples, and 3-dimensional protein modeling. Descriptive statistics, such as frequency counts, were used to describe the distribution of identified pathogenic variants and genotypes. Results: The genetic characteristics of patients with UHS were established in 80 of 107 (74.8%) index patients (82 [76.6%] female) who carried biallelic pathogenic variants in PADI3, TGM3, or TCHH (ie, genes that encode functionally related hair shaft proteins). Molecular genetic findings from 11 of these 80 individuals were previously published. In 76 (71.0%) individuals, the UHS phenotype were associated with pathogenic variants in PADI3. The 2 most commonly observed PADI3 variants account for 73 (48.0%) and 57 (37.5%) of the 152 variant PADI3 alleles in total, respectively. Two individuals carried pathogenic variants in TGM3, and 2 others carried pathogenic variants in TCHH. Haplotype analyses suggested a founder effect for the 4 most commonly observed pathogenic variants in the PADI3 gene. Conclusions and Relevance: This cohort study extends and gives an overview of the genetic variant spectrum of UHS based on molecular genetic analyses of the largest worldwide collective of affected individuals, to our knowledge. Formerly, a diagnosis of UHS could only be made by physical examination of the patient and confirmed by microscopical examination of the hair shaft. The discovery of pathogenic variants in PADI3, TCHH, and TGM3 may open a new avenue for clinicians and affected individuals by introducing molecular diagnostics for UHS.

Fast fragment- and compound-screening pipeline at the Swiss Light Source.

Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported.

Central Carbon Metabolism, Sodium-Motive Electron Transfer, and Ammonium Formation by the Vaginal Pathogen Prevotella bivia.

Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.

A Sodium-Translocating Module Linking Succinate Production to Formation of Membrane Potential in Prevotella bryantii.

Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of the rumen microbiota belong to the family Prevotellaceae, which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na+-translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min-1 mg-1), quinone reduction (490 nmol min-1 mg-1), and fumarate reduction (1,200 nmol min-1 mg-1) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii. Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD+ and succinate. We propose that the regeneration of NAD+ in P. bryantii is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. IMPORTANCE Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella spp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na+-translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii. Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.

AgeR deletion decreases soluble fms-like tyrosine kinase 1 production and improves post-ischemic angiogenesis in uremic mice.

Peripheral arterial disease occurs more frequently and has a worse prognosis in patients with chronic kidney disease (CKD). The receptor for advanced glycation end products (RAGE) is involved in multiple aspects of uremia-associated vasculopathy. Previous data suggest that the RAGE pathway may promote soluble fms-like tyrosine kinase 1 (sFlt1) production, an anti-angiogenic molecule. Thus, we tested the hypothesis that the deletion of AgeR would decrease sFlt1 production and improve post-ischemic revascularization in uremic condition. We used a well-established CKD model (5/6 nephrectomy) in WT and AgeR-/- C57/Bl6 mice. Hindlimb ischemia was induced by femoral artery ligation. Revascularization was evaluated by complementary approaches: ischemic limb retraction, LASCA imagery, and capillary density. The production of sFlt1 was assessed at both RNA and protein levels. After hindlimb ischemia, uremic mice showed slower functional recovery (p < 0.01), decreased reperfusion (p < 0.01), lower capillary density (p = 0.02), and increased circulating sFlt1 levels (p = 0.03). AgeR deletion restored post-ischemic angiogenesis and was protective from sFlt1 increase in uremic mice. These findings show the main role of RAGE in post-ischemic angiogenesis impairment associated with CKD. RAGE may represent a key target for building new therapeutic approaches to improve the outcome of CKD patients with PAD.

Concise Synthesis of 1,4-Benzoquinone-Based Natural Products as Mitochondrial Complex I Substrates and Substrate-Based Inhibitors.

A short, efficient one-step synthesis of 2-methyl-5-(3-methyl-2-butenyl)-1,4-benzoquinone, a natural product from Pyrola media is described. The synthesis is based on a direct late C-H functionalization of the quinone scaffold. The formation of the natural product was confirmed by means of 2D-NMR spectroscopy. Additional derivatives were synthesized and tested alongside the natural product as potential substrate and substrate-based inhibitors of mitochondrial complex I (MCI). The structure-activity relationship study led to the discovery of 3-methylbuteneoxide-1,4-anthraquinone (1 i), an inhibitor with an IC50 of 5 µM against MCI. The identified molecule showed high selectivity for MCI when tested against other quinone-converting enzymes, including succinate dehydrogenase, and the Na (+)-translocating NADH:quinone oxidoreductase. Moreover, the identified inhibitor was also active in cell-based proliferation assays. Therefore, 1 i can be considered as a novel chemical probe for MCI.

Anoxic cell rupture of Prevotella bryantii by high-pressure homogenization protects the Na+-translocating NADH:quinone oxidoreductase from oxidative damage.

Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L-1 culture and a NADH oxidation activity of 0.8 µmol min-1 mg-1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii.

Impact of Na+-Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae.

The Na+ ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF, a flavin attachment gene, apbE, and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δnqr and ΔubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe2+ (Feo), was increased in V. choleraeΔnqr In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. choleraeΔnqr with Fe2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. choleraeΔnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation.IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics.

Cryo-EM structure of a transthyretin-derived amyloid fibril from a patient with hereditary ATTR amyloidosis.

ATTR amyloidosis is one of the worldwide most abundant forms of systemic amyloidosis. The disease is caused by the misfolding of transthyretin protein and the formation of amyloid deposits at different sites within the body. Here, we present a 2.97 Å cryo electron microscopy structure of a fibril purified from the tissue of a patient with hereditary Val30Met ATTR amyloidosis. The fibril consists of a single protofilament that is formed from an N-terminal and a C-terminal fragment of transthyretin. Our structure provides insights into the mechanism of misfolding and implies the formation of an early fibril state from unfolded transthyretin molecules, which upon proteolysis converts into mature ATTR amyloid fibrils.

Respiratory Membrane Protein Complexes Convert Chemical Energy.

The invention of a biological membrane which is used as energy storage system to drive the metabolism of a primordial, unicellular organism represents a key event in the evolution of life. The innovative, underlying principle of this key event is respiration. In respiration, a lipid bilayer with insulating properties is chosen as the site for catalysis of an exergonic redox reaction converting substrates offered from the environment, using the liberated Gibbs free energy (ΔG) for the build-up of an electrochemical H+ (proton motive force, PMF) or Na+ gradient (sodium motive force, SMF) across the lipid bilayer. Very frequently , several redox reactions are performed in a consecutive manner, with the first reaction delivering a product which is used as substrate for the second redox reaction, resulting in a respiratory chain. From today's perspective, the (mostly) unicellular bacteria and archaea seem to be much simpler and less evolved when compared to multicellular eukaryotes. However, they are overwhelmingly complex with regard to the various respiratory chains which permit survival in very different habitats of our planet, utilizing a plethora of substances to drive metabolism. This includes nitrogen, sulfur and carbon compounds which are oxidized or reduced by specialized, respiratory enzymes of bacteria and archaea which lie at the heart of the geochemical N, S and C-cycles. This chapter gives an overview of general principles of microbial respiration considering thermodynamic aspects, chemical reactions and kinetic restraints. The respiratory chains of Escherichia coli and Vibrio cholerae are discussed as models for PMF- versus SMF-generating processes, respectively. We introduce main redox cofactors of microbial respiratory enzymes, and the concept of intra-and interelectron transfer. Since oxygen is an electron acceptor used by many respiratory chains, the formation and removal of toxic oxygen radicals is described. Promising directions of future research are respiratory enzymes as novel bacterial targets, and biotechnological applications relying on respiratory complexes.

Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL amyloidosis.

Amyloid fibrils derived from antibody light chains are key pathogenic agents in systemic AL amyloidosis. They can be deposited in multiple organs but cardiac amyloid is the major risk factor of mortality. Here we report the structure of a λ1 AL amyloid fibril from an explanted human heart at a resolution of 3.3 Å which we determined using cryo-electron microscopy. The fibril core consists of a 91-residue segment presenting an all-beta fold with ten mutagenic changes compared to the germ line. The conformation differs substantially from natively folded light chains: a rotational switch around the intramolecular disulphide bond being the crucial structural rearrangement underlying fibril formation. Our structure provides insight into the mechanism of protein misfolding and the role of patient-specific mutations in pathogenicity.

Receptor for advanced glycation end products: a key molecule in the genesis of chronic kidney disease vascular calcification and a potential modulator of sodium phosphate co-transporter PIT-1 expression.

BACKGROUND: Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, frequent vascular calcification (VC) and accumulation of uraemic toxins. Advanced glycation end products and S100 proteins interact with the receptor for advanced glycation end products (RAGE). In the present work, we aimed to investigate the role(s) of RAGE in the CKD-VC process. METHODS: Apoe-/- or Apoe-/-Ager (RAGE)-/- male mice were assigned to CKD or sham-operated groups. A high-phosphate diet was given to a subgroup of Apoe-/-and Apoe-/-Ager-/- CKD mice. Primary cultures of Ager+/+ and Ager-/- vascular smooth muscle cells (VSMCs) were established and stimulated with either vehicle, inorganic phosphate (Pi) or RAGE ligands (S100A12; 20 µM). RESULTS: After 12 weeks of CKD we observed a significant increase in RAGE ligand (AGE and S100 proteins) concentrations in the serum of CKD Apoe-/- mice. Ager messenger RNA (mRNA) levels were 4-fold higher in CKD vessels of Apoe-/- mice. CKD Apoe-/- but not CKD Apoe-/- or Ager-/- mice displayed a marked increase in the VC surface area. Similar trends were found in the high-phosphate diet condition. mRNA levels of Runx2 significantly increased in the Apoe-/- CKD group. In vitro, stimulation of Ager+/+VSMCs with Pi or S100A12 induced mineralization and osteoblast transformation, and this was inhibited by phosphonoformic acid (Pi co-transporters inhibitor) and Ager deletion. In vivo and in vitro RAGE was necessary for regulation of the expression of Pit-1, at least in part through production of reactive oxygen species. CONCLUSION: RAGE, through the modulation of Pit-1 expression, is a key molecule in the genesis of VC.