Protective / Preventive Role Of Natural Compounds In Human Health: Biochemical Approaches To Target Ageing And Neurodegeneration
01/06/2022 Silvana Hrelia and Cristina Angeloni University of Bologna
The modern “Nutrition 3.0” model will be introduced focusing on the growing interest in nutraceutical bioactive compounds. In particular, the role of biochemical research in identifying the molecular and cellular mechanisms underpinning the protective/preventive role of nutraceutical molecules against aging and neurodegeneration will be considered. Neurodegenerative diseases, such as Alzheimer's and Parkinson’s diseases, are multifactorial disorders characterised by the progressive loss of neurons in the central nervous system. Although each neurodegenerative disease exhibits specific pathological features, they also share some common molecular mechanisms, such as abnormal protein aggregation, mitochondrial disfunction, neuroinflammation and oxidative stress. The lack of effective treatments and the multi-factorial etiology of these disorders has developed a pressing need to identify preventive/therapeutic compounds with pleiotropic activity. Among the many risk factors for neurodegenerative diseases, the aging process has the strongest impact and the investigation on basic mechanisms of aging and their role in the onset and progression of neurodegenerative disease are fundamental to develop effective preventive/therapeutic strategies.
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Mechanisms of ligand binding
27/05/2022 ore 17.00 Enrico Di Cera MD Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis
Ligand binding is at the basis of all biological interactions and has long been subject to theoretical and experimental investigation. The presentation reviews basic mechanisms of ligand binding, from lock and key to induced fit and conformational selection (1). A case is made for the dominance of conformational selection in biology.
1. Di Cera E. Mechanisms of ligand binding. Biophys Rev (2020) 1, 011303.
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NADPH Oxidase: Structure, Enzymology and Drug Design
20/05/2022 Andrea Mattevi Department of Biology and Biotechnology University of Pavia, Italy
NADPH oxidases (NOXs) are the only known human enzymes solely in charge of ROS production. In addition to their roles in the innate immunity and response to stressful conditions, NOXs are part of the redox signaling pathways that sustain cell proliferation, oncoprotein (e.g. RAS) driven cell transformation, and tumor microenvironment manipulation. NOXs are tightly controlled and understanding the molecular mechanisms underlying their isoform-specific regulation is an open issue with far-reaching implications for drug design.
In 2017, our group in Pavia has solved and published the first crystal structures of the dehydrogenase and transmembrane domains of a bacterial NOX5, 40% identical to human NOX5 [1-3]. Computer modelling was then used to view the overall structure of the NOX catalytic core. This landmark result revealed the structural basis for flavin reduction, “across-the-membrane” electron transfer, and ROS generation on the outer side of the membrane. In parallel to the structural studies, we have thoroughly investigated the putative NOX inhibitors that have been reported in the literature. This painstaking project led us to realize that virtually all known NOX inhibitors (31 of them were evaluated) suffer from off-targets effects, such as ROS scavenging and assay interference. This issue is so overwhelming that it was impossible to discern between the non-specific effects exerted by these compounds and their specific binding to NOXs (if any [4]). We have therefore employed the NOX5 dehydrogenase domain as our initial platform to carry on a drug design campaign. To this end, together with our collaborators at Harvard Medical School and Dana Farber Cancer Institute, we have conducted an ultra large-library computational screening using our NOX5 dehydrogenase domain PDB structure. We have evaluated the chosen library using a robust and high throughput workflow comprising primary and orthogonal assays as well as control assays to probe assay interfering compounds and ROS scavengers. Protein crystallography studies of the protein with the best hits have yielded for the first-time a crystal structure of this class of enzymes in complex with our studied inhibitors. Accordingly, our current efforts rely on the study and development of new and effective isoform-specific NOX inhibitors and the understanding of their effect on cancer model cells in which NOXs have a key role.
[1] Magnani, F., Nenci, S., Millana Fananas, E., Ceccon, M., Romero, E., Fraaije, M.W., Mattevi*, A. (2017) Crystal structures and atomic model of NADPH oxidase. Proc. Natl. Acad. Sci. USA 114, 6764-6769.
[2] Oosterheert, W., Reis, J., Gros, P., Mattevi, A. (2020) An Elegant Four-Helical Fold in NOX and STEAP Enzymes Facilitates Electron Transport across Biomembranes-Similar Vehicle, Different Destination. Acc. Chem. Res. 53, 1969-1980.
[3] Magnani, F., Mattevi*, A. (2019) Structure and mechanisms of ROS generation by NADPH oxidases. Curr. Opin. Struct. Biol.59, 91-97.
[4] Reis J, Massari M, Marchese S, Ceccon M, Aalbers FS, Corana F, Valente S, Mai A, Magnani F, Mattevi, A. (2020) A closer look into NADPH oxidase inhibitors: Validation and insight into their mechanism of action. Redox Biol. 32, 101466.
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When viral RNA met the cell: a story of protein-RNA interactions
12/05/2022 11 am Alfredo Castello MRC-University of Glasgow Centre for Virus Research (UK)
RNA is a central molecule for the RNA virus life cycle as it functions not only as messenger for the synthesis of proteins, but also as storage of genetic information as genome. Given the central role of viral RNAs in infection, I hypothesised that it must be a hub for critical host-virus interactions. To test this, my laboratory has developed new approaches to elucidate the composition of viral ribonucleoproteins using Sindbis virus, SARS-CoV-2 and human immunodeficiency virus (HIV) as discovery models. With them, we have discovered a new universe of host-virus interactions with central regulatory roles in infection. Interestingly, these viruses, despite having different sequences and infection cycles, engage with a largely shared pool of cellular RNA-binding proteins. The efforts of my laboratory are now focused on understanding the molecular mechanisms underpinning these master regulators of virus infection, as we envision that they are promising targets for broad-spectrum antiviral therapies.
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RNA-based therapeutic approaches for neurodegenerative tauopathies
06/05/2022 Michela A. Denti University of Trento
There are currently no disease-modifying therapies for the treatment of tauopathies, a group of progressive neurodegenerative disorders that are pathologically defined by the presence of tau protein aggregates in the brain.
In Frontotemporal Lobar Degeneration (FTLD), a familial tauopathy, 57 different mutations in the microtubule-associated protein tau (MAPT) gene have been identified. About half of these mutations perturb the finely regulated balance in the splicing of MAPT exon 10, inducing the increase of exon 10-containing splicing isoforms. In turn, this affects the number of microtubule-binding domains present in the mature tau protein, ultimately causing neurodegeneration and tau aggregates.
RNA Therapies are recently gaining their momentum and 12 approved RNA-based drugs have been approved as of today.
We have designed three different RNA-based therapeutical approaches for FTLD, which might be beneficial also for other tauopathies: exon-skipping antisense oligonucleotides (AONs), AAV-vectored antisense chimeric U1 or U7 snRNAs and isoform-specific short interfering RNAs (siRNAs). Via reporter minigenes we have screened several of these molecules. We have validated the efficacy of the best hits for each approach in cultured cells. We are presently performing Proof-of-Concept studies in two orthogonal models of FTLD disease: human iPSc-derived neurons bearing the IVS10+16 mutation and a mouse model bearing the human MAPT gene with the same mutation and recapitulating the disease’s molecular, histopathological and behavioral aspects.
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Metabolic control of T cell immunity
29/04/2022 Dott. Mauro Corrado, CECAD center, University of Cologne (Germany)
Tissue homeostasis is maintained via a fine balance between pro- and anti-inflammatory signals, a balance that is lost when mitochondrial metabolism is compromised in T cells, resulting in impaired immunity, multimorbidity and inflammation.
The dynamic metabolism of T cells during an infection and upon nutrient stress depends indeed on the ability of T cells to fine tune their mitochondrial lipid composition (and in particular their content of the specific mitochondrial lipid cardiolipin).
Nevertheless, how different tissue specific microenvironments and immune cells crosstalk and determine the damage to different tissues as well as the systemic outcome of a metabolic dysfunction in T cell is poorly understood.
Our lab combines in vivo mouse phenotyping, classic immunology and biochemistry with quantitative proteomics and metabolomics to study the immune response in mouse models and patients affected by mitochondrial diseases.
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Architecture of the human erythrocyte ankyrin-1 complex
22/04/2022 ORE 17.00 Francesca Vallese Dept. of Anesthesiology Columbia University, New York, USA
The ankyrin-1 complex tethers the spectrin-actin cytoskeleton to the red blood cell (RBC) membrane, and acts as a metabolic hub, connecting membrane proteins that are involved in gas exchange, pH control, and regulation of cellular volume and deformability. Mutations in components of the complex lead to inherited defects in erythrocyte shape and stability, such as hereditary spherocytosis. Ankyrins are also broadly expressed adaptors functioning as master-organizers of membrane-associated protein complexes in neurons and other cell types. However, the precise composition of the ankyrin-1 complex, and the structural basis for membrane association and recruitment of target membrane proteins remains unknown in any context. We solve the single particle cryo-EM structures of the human ankyrin-1 complex. Our structures reveal the architecture of this 1.2 MDa supercomplex, which includes the Rhesus polypeptides RhCE & RhAG, ankyrin 1, protein 4.2 and three copies of the dimeric band 3 anion exchanger bound to glycophorin A, assembled into a 1.2 MDa supercomplex. Additional complexes carrying one aquaporin-1 (AQP1) tetramer are also identified. The structure of membrane-bound ankyrin shows that the first five repeats adopt an unexpected T-shaped configuration whereby the inner groove is oriented parallel to the membrane, facilitating recognition of integral membrane binding partners such as RhCE and AQP1. Both the inner groove and the convex outer surface of ankyrin participate in specific interactions with protein 4.2 and band 3. Our structures also reveal the architecture and stoichiometry of the heterotrimeric Rh channel, highlighting the role of ankyrin in mediating clustering of multiple copies of structurally diverse membrane proteins.
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Evolution of Klebsiella pneumoniae high-risk clones to pan-resistance
08/04/2022 Alessandra Carattoli Dept. Molecular Medicine Sapienza University of Rome
Infections caused by antibiotic-resistant Klebsiella pneumoniae are considered a major emerging problem in hospitals. K. pneumoniae behaves like an opportunistic bacterium, causing serious infections especially in the most fragile and long-term patients. In the nosocomial field, the greater burden of infections is due to the global dispersion of some high-risk clones of K. pneumoniae that spread successfully in Asia, the United States, Europe.
Genomics made possible to analyze and understand the evolutionary strategies of these successful clones and their adaptation to antimicrobial use, leading to the emergence of clinically relevant mechanisms of resistance to antibiotics. Since the third generation cephalosporin resistance in the early 2000s, K. pneumoniae has progressively evolved acquiring resistance to carbapenems, colistin, tigecycline and more recently fosfomycin and new drug-inhibitor combinations such as ceftazidime-avibactam in the last decade. Pan-resistant clones (defined as cells resistant to all classes of antibiotics) and treatable only with the newest formulation drugs have been reported in the world and could quickly represent a microbiological problem of strategic relevance in clinical settings.
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Mechanism of a cell wall transporter involved in lipoteichoic acid synthesis and bacterial adaptation
01/04/2022 Camilo Perez. Biozentrum, Basel, CH
Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters take part in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signalling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. We elucidated the structure of LtaA, and showed that it functions as a proton-dependent lipid antiporter, which contributes to the adaptation of the pathogen Staphylococcus aureus to acidic conditions, common in the skin and nasopharynx of the human host. LtaA takes part in the assembly of lipoteichoic acid, important for protection of S. aureus from environmental stress, host cell adhesion, antibiotic resistance, and immune evasion. The essential role of LtaA in adjusting the pool of glycolipids available for lipoteichoic acid assembly makes it a potential target for drugs aiming to counteract antimicrobial-resistant S. aureus strains e.g., methicillin-resistance S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). Recently, we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA. We revealed that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a ‘trap-and-flip’ mechanism that might be shared among MFS lipid transporters.
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Amino acid metabolism: involvement in tumor progression and new therapeutic approaches
25/03/2022 Dott. Alessio Paone (Dept. Biochemical Sciences, Sapienza University of Rome)
Metabolic adaptation is necessary for malignant tumor cells to survive the extreme conditions to which they are exposed within the mass of the primary tumor as well as throughout the metastatic process. In recent years it has been described that, despite having all the biosynthetic pathways intact, cancer cells of different origins are dependent on extracellular non-essential amino acids to support fundamental processes such as proliferation and survival. Here I will present data showing the centrality of amino acid metabolism, in particular of one-carbon metabolism, in the survival and metastatic ability of lung cancer cells. We studied serine hydroxymethyl transferases (SHMT), proteins fundamental in serine/glycine metabolism and revealed peculiar new properties such as their RNA binding ability. Taking advantage of these results, we are developing new approaches based on small RNA molecules to inhibit SHMTs, an enzyme for which effective small molecule inhibitors do not yet exist. We are also studying how the availability of selected amino acids (serine/glycine/glutamate) affect the ability of highly metastatic lung and breast cancer cells to select the brain as a target organ for metastasis formation. We clarified that these cells exploit extracellular amino acids to increase their extravasation ability, and that interfering with the import of these amino acids, which are abundant in the brain parenchyma, limits the formation of metastases in this organ, suggesting a novel therapeutic strategy to limit brain metastasis formation, a major cause of death in cancer patients.
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