Seminars


Every year, the PhD school organizes or contribute to organize seminars on topics related to the PhD curricula.
In this page, we also alert students for seminars on related topics.

The 2022 link to connect is

https://uniroma1.zoom.us/j/81855430354?pwd=U1VqWHZvL2haYWVhZHdXRHFrTzNQZz09
Passcode 582881

2022


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.
Mechanisms of ligand binding
27/05/2022 27/05/2022 at 5 pm 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.
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.
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.
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.
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.
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.
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.
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.
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.
Functional and Pathological Interactions of α synuclein
18/03/2022 Alfonso De Simone Department of Pharmacy, University of Naples Federico II
The aggregation of α-synuclein (αS), a neuronal protein that is abundant at the pre-synaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions including Parkinson’s Disease (PD). Fibrillar aggregates of αS are the major constituents of proteinacious inclusions known as Lewy bodies that form in dopaminergic neurons of patients suffering from these conditions. The function of αS, however, is currently unknown, with evidences suggesting a role in the regulation of the trafficking of synaptic vesicles. We study the structure and interactions of αS in its functional state and in the form of pathological aggregates by means of biophysics and biomolecular NMR [1]. Our research has identified the nature of the physiological membrane interaction of αS and elucidated how this transient binding is involved functional processes such as the clustering of synaptic vesicles [2] or their docking onto the plasma membrane [3]. In the context of αS aggregation, we focus on the properties of elusive intermediate oligomers and how they impair neuronal function in the context of PD [4-5]. References 1. Fusco G, et al, (2014) Nat Commun 5:3827. 2. Man WK, et al, (2021) Nat Commun 12:927. 3. Fusco G, et al, (2016) Nat Commun 7:12563. 4. Fusco G, et al, (2017) Science 358:1440-3. 5. Cascella, et al, (2019) ACS chem bio 14:1352-1362.
Emerging role for m6A RNA modification in cancer: learning from leukemia
11/03/2022 Alessandro Fatica Dept. Biology and Biotechnology Sapienza
We are currently assisting at the explosion of the epitranscriptomics, which studies the functional role of chemical modifications into RNA molecules. Among more than 100 RNA modifications, the N6-methyladenosine (m6A) has attracted the interest of researchers all around the world. m6A is the most abundant internal chemical modification in mRNA and it can control any aspect of mRNA post-transcriptional regulation. m6A is installed by “writers”, removed by “erasers”, and recognized by “readers”, thus, it can be compared to the reversible and dynamic epigenetic modifications in histones and DNA. Given its fundamental role in determining the fate of mRNAs, it comes as no surprise that alterations to m6A modifications have a deep impact in cell differentiation, normal development and human diseases. Here, I will present the important role of m6A modification in gene expression and its contribution to cancer development. In particular, I will focus on myeloid leukaemia that, among first, has indicated how alteration in m6A modification can disrupt normal cellular proliferation and lead to cancer.

2021


Oxidative Stress: Biochemical and Pharmacological Aspects
Starting 11/02/2021 - multiple dates
https://antiox.it/programme
Search for efficient diagnosis and therapy of advanced melanoma using biophysical methods
28/10/2021 at noon Prof. Tomasz Kobiela Warsaw University of Technology, Head of Laboratory of Biomolecular Interactions Studies
Melanoma, which originates from melanocytes, after entering the metastatic stage causes the highest mortality among skin malignant tumors resulting from the lack of effective therapy due to common resistance to the applied drugs [1]. In the view of increasing worldwide prevalence the intensive search for successful therapy pattern is undertaken with the use of combined strategy. This includes the combination of recognized anti-cancer drugs with compounds acting on various vital cellular signaling pathways, particularly on the energy metabolism. In melanoma, the mutation of the proto-oncogene B-Raf cytoplasmic serine–threonine kinase (BRAF) gene is the most common (over 50% of patients), which is why most of the applied drugs are directed at inhibiting that signaling pathway. For the optimization of the choice of possible compounds, we performed the modified SynGeNet drug combination prediction study [2]. Experimental validation of effective combinations was done by monitoring the interaction of specific lectins with cellular surface glycans typical for various stages of melanoma progression in real-time experiments using quartz crystal microbalance with the dissipation monitoring and atomic force microscopy [3]. Evaluation of the modification of the glycosylation process of metastatic melanoma cells as result of the applied combination of compounds could reveal potential usability in malignant melanoma treatment. [1] Sobiepanek, A. et al. (2021). European Biophysics Journal, 50, 523. [2] Regan-Fendt, K. E. et al. (2019). Npj Systems Biology and Applications, 5(1), 6. [3] Sobiepanek et al. (2017) Biosensors and Bioelectronics, 93:274-281
Structural Basis for SARS-CoV-2 Neutralization by Human Antibodies
18/06/2021 15.00 Dr. Gabriele Cerutti, Zuckerman Institute Columbia University, New York, USA
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19, emerged in late 2019, rapidly establishing an ongoing worldwide pandemic with more than a hundred million infected and over three million dead. In response, an unprecedented global effort to develop vaccines and therapeutics is well underway. One promising approach is the identification and structural characterization of SARS-CoV-2-neutralizing antibodies, which could be used as therapeutic or prophylactic agents. Potent neutralizing antibodies directed against SARS-CoV-2 spike, isolated from infected patients, target two main regions: the receptor binding domain (RBD) and the N-terminal domain (NTD). RBD-directed neutralizing antibodies target different epitopes on the domain and neutralize the virus by blocking receptor binding. NTD-directed neutralizing antibodies target a single supersite and their mechanism of action is less clear. Circulating SARS-CoV-2 variants seem to arise in response to human antibody pressure and the molecular basis for immune evasion or accommodation of mutants can be explained from a structural perspective.
Transcription factor EB in vascular and heart biology
04/06/2021, Prof. Federico Bussolino, Department of Oncology, University of Torino Candiolo Cancer Institute-IRCCS-FPO
TFEB (Transcription factor EB) represents an emerging player in the biology of cardiovascular system. TFEB was originally described to be translocated in a juvenile subset of paediatric renal cell carcinoma but whole genome sequencing reported somatic mutations sporadically found in many different cancers. Besides its oncogenic activity, TFEB controls the autophagy-lysosomal pathway by recognizing a recurrent motif present in the promoter regions of a set of genes that participate to lysosome biogenesis and its dysregulation is instrumental in the pathogenesis of lysosomal storage diseases. Emerging findings suggest that TFEB exerts wider regulatory activities in response to stress encompassing immunity, longevity and metabolism. In this seminar I’ll summarize the data of my Lab obtained by the specific deletion of Tfeb in mouse endothelial and epicardial cells. Our data demonstrate that TFEB activates specific genetic programs respectively regulating cell-cycle in endothelial cells and epithelial-mesenchymal transition in epicardial cells. Recognizing vascular and epicardial TFEB as a hub of a network of signals between tissues and bloodstream provides a fresh perspective on the molecular principles regulating organogenesis and tissue functions in physiology and pathology.
Molecular nature and regulation of the mitochondrial permeability transition pore
28/05/2021 Paolo Bernardi, Department of Biomedical Sciences and CNR Neuroscience Institute, University of Padova, Italy
Molecular nature and regulation of the mitochondrial permeability transition pore Paolo Bernardi* Department of Biomedical Sciences and CNR Neuroscience Institute, University of Padova, Italy The molecular nature of the mitochondrial permeability transition (PT) is a long-standing mystery of mitochondrial biology. Occurrence of the PT strictly requires matrix Ca2+ accumulation and is favored by matrix cyclophilin D, which mediates the inhibitory effects of cyclosporin A. Several hypotheses about the molecular nature of the PT have been put forward over the years. Today the prevailing view is that permeabilization of the inner membrane follows opening of a high-conductance channel, the PT pore, which is also called mitochondrial megachannel or multiconductance channel. I will provide an overview of the field with specific emphasis on the potential role of F-ATP synthase in channel formation.
The evolving spectrum of vitamin B6 responsive disorders
21/05/2021 Philippa Mills Great Ormond Street Institute of Child Health, University College London, London, UK.
Vitamin B6 is a micronutrient essential for normal physiological functioning in humans, serving as a cofactor for >70 human enzymes. Unlike lower organisms, humans cannot synthesise this vitamin and rely on dietary forms and those synthesised by the microbiome. These dietary forms include pyridoxine, pyridoxamine and pyridoxal and their 5’-phosphorylated derivatives. The intestine can only absorb the non-phosphorylated B6 vitamers, therefore phosphorylated forms must first be hydrolysed. Specific enzymes are then involved in the conversion of pyridoxine, pyridoxamine and pyridoxal to pyridoxal 5’-phosphate (PLP), the only active form of vitamin B6. Inborn errors of metabolism which affect the interconversion and availability of PLP, lead to a deficiency of this vitamer which manifests as an epilepsy that responds to treatment with supraphysiological doses of vitamin B6. This is not surprising given the vital role that PLP plays in neurotransmitter metabolism. Disorders include pyridox(am)ine phosphate oxidase deficiency (a disorder affecting PLP synthesis and recycling), disorders where metabolites accumulate that inactivate PLP, for example, ALDH7A1 deficiency and hyperprolinaemia type II, disorders which affect PLP import into the brain (hypophosphatasia and glycosylphosphatidylinositol anchor synthesis defects), and the recently described disorder in which mutations in an intracellular PLP-binding protein result in abnormal B6 homeostasis (PLPHP deficiency). We have shown recently however, by using next generation sequencing for the investigation of patients that have remained undiagnosed for many years, that: i) disorders of vitamin B6 deficiency do not always manifest as an epilepsy ii) supraphysiological doses of B6 can not only be used to treat the vitamin B6-dependent epilepsy disorders but also other seizure disorders that are not associated with vitamin B6 metabolism, such as the KCNQ2-related epileptic encephalopathies.
Diet and Metabolic Therapeutics in Cancer
14/05/2021 Jason LoCasale (Duke University School of Medicine, USA).
This presentation will discuss efforts to understand glucose and amino acid metabolism in cancer biology using metabolomics approaches. First, I will discuss new work on our understanding of central carbon metabolism. This part of the talk will focus on efforts to target cancer metabolism by disrupting the processing of macronutrient sources. Several examples of biological consequences of this pathway will then be presented. Next, I will focus on methionine metabolism. I will discuss work on dietary influences on the activity of the pathway and its relation to the regulation of one carbon metabolism in health. How methionine restricted diets may allow for interventions in cancer treatment will also be discussed. This concept also provides a link between nutrient status and chromatin biology which I will briefly touch upon.
“Identification of novel mechanisms in innate antiviral immunity”
07/05/2021 Søren Riis Paludan (Arhus Univ., DK)
The innate immune system is essential for host defense against virus infections. Type I interferon (IFN) is particularly important for early antiviral defense, and understanding of how IFN is induced is therefore of central importance. However, IFN also possesses inflammatory activities, and can contribute to disease development if produced in high amounts or over long time. Therefore, immune mechanisms that control virus infections without inducing IFN expression may contribute to a “silent” layer of the immune system. In this presentation, I will present our recent research on how herpes simplex virus induces expression of type I IFN, and also on novel constitutive immune mechanisms that control the virus.
Rett Syndrome as a model to study neurobiology and candidate therapeutics for brain disorders
30/04/2021 Daniela Tropea Trinity College (Dublin, Ireland)
Rett Syndrome (RTT) is a neurodevelopmental disorder associated to mutations in the X-linked gene MECP2, which codes for the protein Methyl-CpG binding protein 2. MeCP2 acts mainly a chromatin-binding protein, and determines activation or inhibition of gene expression, depending on its binding co-factors. Mecp2 is also involved in RNA splicing. Although mostly associated to RTT, MECP2 is involved in several other neuropsychiatric and neurological conditions, and its dysregulation (both upregulation and downregulation) has functional consequences. In addition, several cellular phenotypes identified in RTT are present in other brain disorders and treatment that benefit patients with RTT are also effective in other neurodevelopmental disorders. In this seminar I will present the evidence that Mecp2 controls many molecular mechanisms across different brain pathologies and that RTT can be used as a model to uncover the pathophysiology of several disorders.
The stressed synapses
23/04/2021 Tiziana Borsello, Università degli Studi di Milano
Synaptopathy define key features of many of neurodegenerative and psychiatric disorders. The synaptic degeneration, which undergo to a first reversible phase of “spine dysfuncion” leading to second irreversible phase of synaptic loss/death, which will lead to dendrite retraction and progress with neuronal death. Because synaptic injury precedes neuronal death and dysfunctional synapse possess a remarkable capacity for repair and functional recovery, we focus our efforts to develop a strategy to protect synapses during this early phase of the brain pathology. However, the intracellular mechanisms regulating synaptic dysfunction in AD are currently not fully understood. We identify the c-Jun N-terminal kinase (JNK) as a key player in AD synaptopathy. We characterized the JNK role in A oligomer-induced synaptopathy in vitro and in vivo in post-synaptic element describing the PSD alteration mediated by JNK. Our results demonstrated that the specific JNK inhibition (D-JNKI1) protected synapsed degeneration. Importantly JNK role in the post synaptic element is double: phophorylation of PSD-95 and Tau. JNK action on PSD-95 induced it down-relulation and PSD marker alterations, while JNK hyper-phosphorylation of Tau, altered Tau and Drebrin interaction, this leads to spine dysmorphogenesis. We then focused on JNK presynaptic localization and its role at this site. We proved that presynaptic fractions contained significant amount of JNK protein and its activated form. With biochemical approaches we demonstrated the interaction between JNK Synataxin-1,2 and Snap25. We defined JNK action on the SNARE complex formation and its role in modulation of vesicle release. These data showed JNK important functional role in post and pre-synaptic element. In conclusion, all together these results set the basis to develop a JNK-base therapeutic strategy to tackle synapse degeneration.
Understanding how RNA-based mechanisms control genome stability in cancer
16/04/2021 Lovorka Stojic - Barts Cancer Institute, Queen Mary University of London, London, UK
Genome stability is paramount to cellular homeostasis throughout the human lifespan. Cells have developed several surveillance mechanisms to protect the genome from mutations and ensure faithful duplication and transmission of the genetic material. Defects in any of these mechanisms leads to genome instability, which drives cancer evolution and contributes to tumour heterogeneity, drug resistance and poor prognosis. Protein-mediated mechanisms controlling genome stability are well described, however, the biological and regulatory function of RNA-based mechanisms in this context, and in particular the contribution of long noncoding RNAs (lncRNAs), is largely unknown. We have recently identified novel lncRNAs linked to chromosome mis-segregation, a process common to different types of cancer. I will focus on two nuclear localised lncRNAs whose expression is altered in cancer, and highlight mechanisms through which these lncRNAs safeguard genome integrity and their relevance to cancer.
Metabolic rewiring driving metastasis formation
09/04/2021 Sarah Maria Fendt (VIB-KU Leuven Center Cancer Biology)
Metabolic rewiring is a hallmark of cancer cells. However, how nutrients drive the ability of cancer cells to rewire their metabolism is poorly defined. We are investigating the in vivo nutrient metabolism during metastasis formation to mechanistically understand how nutrients from the microenvironment enable cancers to progress from a local to a systemic disease. Using 13C tracer infusions in mouse models we find that nutrient availability shapes the metabolism and phenotype of cells and subsequently promotes the progression of cancer. Consequently, interfering with nutrient metabolism emerges as a promising therapeutic strategy against cancer. Taken together, our research highlights that nutrient metabolism is an important driver of cancer progression.
RNA regulates Glycolysis and Embryonic Stem Cell Differentiation via Enolase 1
26/03/2021 Ina Huppertz, European Molecular Biology Laboratory (EMBL)
Metabolic pathways are transpiring to be significant regulatory sites that participate in controlling stem cell fates. One common feature in stem cell differentiation is the metabolic remodelling from aerobic glycolysis to respiration during the exit from pluripotency, with distinct paths taken by different germ layers. Since glycolytic enzymes have been reproducibly found to associate with RNA, we studied the conserved interaction of enolase 1 (ENO1) with RNA in vitro, in human cells, and during mouse embryonic stem cell differentiation. We show that ENO1 specifically binds RNA targets in human and murine cells. RNA ligands inhibit ENO1’s enzymatic activity in vitro, and ENO1’s enzymatic substrates specifically compete with its RNA binding. Increasing the concentration of RNA ligands in cultured cells inhibits glycolysis. We demonstrate that the differentiation of embryonic stem cells to specific germ layers involves changes in ENO1’s RNA binding. Importantly, pluripotent stem cells expressing an ENO1 mutant that is hyper-inhibited by RNA are severely impaired in their glycolytic capacity and in endodermal differentiation, whereas cells with an RNA binding-deficient ENO1 mutant display disproportionately high endodermal marker expression. The data uncover ENO1 riboregulation as a novel form of metabolic control. They also describe an unprecedented mechanism underlying the regulation of stem cell differentiation.
“Single particle cryo-electron microscopy, a quantum leap in structural biology”
19/03/2021 Martino Bolognesi (Dipartimento di Bioscienze e Centro di Ricerca Pediatrica Invernizzi Università di Milano)
The development of direct electron detectors, coupled to cryo-vitrification methods and developments of computational approaches, brought to the explosion of the “structural revolution” just a few years ago. Based on the above developments it is now possible to solve the 3D structure of macromolecular aggregates, protein:protein and protein:nucleic acid complexes in an almost direct approach, often reaching near atomic resolution. A real explosion of new structures and studies on macromolecular complexes is currently witnessed throughout the literature. In particular, it must be noted that specific macromolecular samples that could not be faced by previous techniques (e.g amyloid fibrils) are now within reach, shedding first light and entirely new studies on crucial research lines. I will present an overview of the single particle cryoEM method, explaining the basic principles behind, on one hand, and the practical lab aspects involved in sample/grid preparation, on the other. Following the theoretical aspects, I will briefly present the results of three research lines from our lab, focusing on the experimental approaches followed and on the biological implications of the results achieved.
“Analysis of protein-protein complexes and scoring of docking models”
12/03/2021 Romina Oliva (Dipartimento di Scienze e Tecnologie, Università di Napoli “Parthenope”Università Parthenope, Napoli)
Most proteins fulfill their functions through interaction with other proteins. A detailed structural analysis of the interaction between proteins in functional complexes is fundamental for understanding the mechanisms underlying biological processes and for possible biomedical and biotechnological applications. However, a dramatic disproportion still exists between the number of experimental structures solved for protein complexes and the number of structures available for single proteins. In this scenario, molecular docking, i.e. predicting the structure of a protein complex starting from the two separate components, is the method of choice for investigating the molecular basis of recognition in many functional biological systems. Reliably predicting the three-dimensional structure of protein-protein complexes by molecular docking is nonetheless an open challenge, with one of the critical steps being the scoring, i.e. the ability to discriminate between correct and incorrect solutions within a wide pool of generated models. In the last 10 years we have developed tools both for the analysis of the interface in biomolecular complexes and for the scoring of protein-protein docking models. Since 2013, our scoring algorithms have been blindly tested in CAPRI (Critical Assessment of PRedicted Interactions) experiments, docking challenges launched worldwide, where they proved to perform competitively with the state-of-the-art methods in the field.
“Templated Folding of Intrinsically Disordered Proteins”
05/03/2021 Angelo Toto (Department of Biochemical Sciences (Sapienza University)
Our understanding of protein biochemistry is mostly based on the structure-function relationship. This approach was revolutionized by the discovery that proteins can lack a well defined three dimensional structure, while being able to exert their functions in the cellular environment. These proteins are defined as Intrinsically Disordered Proteins, or IDPs. Since the discovery of protein disorder, IDPs have been extensively studied, and for many of them a disorder-to-order transition has been reported upon binding with their natural ligands. By focusing on the interaction between the transactivation domain of the cMyb protein, a prototypical IDP, and the globular KIX domain of the CBP protein, the fundamentals of the methodology used to study binding induced folding reactions will be recapitulated, together with the biophysical approach to interpret data. In particular it will be shown that the folding pathway of cMyb is malleable and dictated by the binding partner KIX. This mechanism is called “templated folding” and we propose it to be a general folding mechanism for IDPs.

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