Seminars







March 1 2024. Every year the Doctorate organizes or participates in the organization of seminars held by Italian and foreign teachers on interesting topics. The seminars are held in person or remotely (on the dates indicated). On this page you will find reports relating to these seminars and those of interest on similar topics.


In 2024 the seminars will be held in room D of "Medicina Legale" (Building CU023) at 2 pm

Prof. Maria Luisa Mangoni & Prof. Francesco Malatesta



The seminars will also be held online at: https://meet.google.com/yuq-idoi-bzx

 

2024


Dr. Stephen Hawser. CEO IHMA Europe Sàrl Monthey , Swit
May 24, 2024 - 2:00 pm
Antimicrobial resistance (AMR) continues to evolve and spread beyond all boundaries. As a result, many infections have become more challenging or even impossible to treat, leading to an increase in morbidity and mortality. Despite the failure of conventional, traditional antimicrobial therapy, in the past two decades, no novel class of antibiotics has been introduced. While the COVID-19 pandemic was frequently mentioned daily worldwide, the AMR crisis is rarely talked about, hence it being labelled as the “Silent Pandemic”. Yet, there are more infections and deaths per year attributed to AMR compared with COVID-19 and indeed many COVID-19 patients died from antimicrobial resistant infections. Currently there are new therapeutic options, especially combination therapies though they are not completely novel. These therapies are helping to slow the silent pandemic but inevitably resistance is emerging. If no new therapies or technologies are developed for the future, AMR will be unstoppable and mortality rates will significantly increase. Fortunately, novel molecules amongst others are in early to mid-stage development though it remains to be seen if these can be developed quickly enough in order to be of clinical utility.
Prof. Maurizio Simmaco. The pivotal role of functional biochemistry in precision medicine
May 17, 2024 - 2:00 pm
The number of people aged 65 years or more in the EU is expected to grow from around 84 million in 2008 to around 141 million by 2050 and they will account for 28.7% of the EU-28’s population by 2080. Italy is in second place in Europe by old age index, with strong consequences on health care system, due to the high number of chronically ill people. There is an urgent need for implementation in standard clinical practice of novel methods to ensure efficacy and safety of polypharmacy in the elderly. Precision Medicine (PM) represent a patient-centered model of health care, based on preemptive and monitoring strategies developed in the last decade thanks to the availability of novel cost-effective technologies to achieve deep molecular characterization of both patients and diseases. The striking impact of the proposed strategy derives by the system-level character of the project, intended to integrate a novel model of healthcare in an Italian National Health System (INHS) structure, that is the academic hospital Sant’Andrea of Rome. Such unique setting allow to integrate sophisticated preventive and monitoring strategies in the usual clinical workflow. The data constitute a proof-of-principle concerning the effectiveness of integration of PM in current clinical practice to improve adherence to therapies and clinical outcomes and is expected to provide evidence that translation of PM into the INHS is indeed actionable.
Prof. Barbara Zambelli. Measuring heat to understanding catalysis: isothermal titration
May 10, 2024 - 2:00 pm
Isothermal titration calorimetry (ITC) is a widely used technique to characterize the thermodynamics of biochemical reactions, as it uses the heat released or absorbed upon virtually all reactions as an internal probe. Indeed, the measured heat is related to the amount of reacting molecules and to the rate of the reaction of interest. The ability of this technique to derive the thermodynamic parameters of the biochemical processes makes it an invaluable and powerful technique to study biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, ΔH) of enzymatic reactions. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. These properties render ITC a unique tool to study enzyme kinetics in applications such as drug discovery. These features will be described and discussed together with some applications to specific cases of enzyme catalysis and inhibition. A general guideline to choose the right procedure according to the system under analysis is given, together with some experimental tips on how to adjust the conditions for increased data quality. The method to analyze the obtained raw ITC curves and to derive the kinetic parameters and inhibition constants is described.
Prof. Oliver Einsle - Unifying Concepts in Nitrogenase Catalysis
May 3, 2024
In the talk, the mechanism of nitrogenase will be discussed. Nitrogenases are responsible for the reduction of nitrogen (N2) to ammonia (NH3). Nitrogenases are the only family of enzymes known to catalyze this reaction, a step in the process of nitrogen fixation, in which the triple bond of the inert N2 molecule is broken. In the seminar, features of and problems of biological nitrogen fixation will also be introduced.
Prof. Cláudio M. Gomes. Faculdade de Ciências da Unive
April 19, 2024 - 2:00 pm
In this seminar I will provide an overview of how defective proteostasis underlies various neurodegenerative diseases characterized by protein deposition. I will unveil our recent discoveries demonstrating the neuroprotective functions of brain proteins from the S100 family. These proteins act as intra and extracellular chaperones, effectively halting the self-assembly and neurotoxicity of tau and amyloid β aggregates. I will delve into the underlying mechanisms of action, particularly focusing on S100B, and will conclude by proposing the hypothesis that S100 proteins collectively form a novel type of protective chaperone network in the nervous system. 1. Hagmeyer et al (2019) Frontiers in Neuroscience https://10.3389/fnins.2019.00640 2. Cristóvão et al (2018) Science Advances https://doi.org/10.1126/sciadv.aaq1702 3. Moreira et al (2021) Nature Communications https://10.1038/s41467-021-26584-2 4. Figueira et al (2022) Journal Molecular Biology https://doi.org/10.1016/j.jmb.2022.167791 5. Moreira and Gomes (2023) Journal Neurochemistry https://doi.org/10.1111/jnc.15756 6. Figueira et al (2023) S100B Front. Neuroscience https://doi.org/10.3389/fnins.2023.1162741
Dr. Michele Fiore. Following ATP route in biological system
March 22, 2024 - 2.00 pm
Adenosine triphosphate (ATP) is one of the most important nucleotides. Found in all known life forms, it is often called the "molecular unit of currency" of intracellular energy transfer. It provides energy to drive and support many processes in living cells, such as muscle contraction, nerve impulse propagation, condensate dissolution, and chemical synthesis. In recent years, and collaboration with different research groups (Brazil, France, Japan, Italy) we have started a deep interest in the analysis of extracellular ATP to monitor such diseases where the degradation of this nucleotide is the substrate for the tissue non-specific alkaline phosphatase (TNAP), suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular ATP is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. Degradation of ATP and mineralization by tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phosphate (Pi) in the early stages of Matrix Vesicles-mediated mineralization. Thus, we have studied with different chemical-physical techniques the degradation of APT in forming PPi: 31P NMR, IR. Our findings suggest that high ATP levels released by cells near vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.
Prof. Michele Vendruscolo: Yusuf Hamied Department of Chemistry University of Cambridge, UK.Thermodynamic and kinetic approaches for drug discovery to target protein misfolding and aggregation
March 8, 2024 - 2:00 pm
Protein misfolding diseases, including Alzheimer’s and Parkinson’s diseases, are characterised by the aberrant aggregation of proteins. These conditions are still largely untreatable, despite having a major impact on our healthcare systems and societies. To address this problem, I will describe drug discovery strategies to target protein misfolding and aggregation. More specifically, I will compare thermodynamic approaches based on the stabilization of the native states of proteins with kinetic approaches based on the slowing down of the aggregation process. This comparison will be carried out in terms of the current knowledge on the process of protein misfolding and aggregation, the mechanisms of disease and the therapeutic targets.
Dr. Micol Falabella: Department of Neuromuscular Diseases University College London (UK)..The role of cardiolipin in mitochondrial disorders
March 1, 2024 - 3:00 pm
Primary mitochondrial diseases (PMDs) are among the most common inherited neurological disorders. They are caused by pathogenic variants in mitochondrial or nuclear DNA that disrupt mitochondrial structure and/or function, leading to impaired oxidative phosphorylation. One emerging subcategory of PMDs involves defective phospholipid (PL) metabolism. Cardiolipin (CL), the signature PL of mitochondria, resides primarily in the inner mitochondrial membrane, where it is biosynthesised and remodelled via multiple enzymes and is fundamental to several aspects of mitochondrial biology. Disruption to genes involved in CL biosynthesis and remodelling has recently been associated with PMD. However, the pathophysiological mechanisms that underpin human CL-related disorders are not fully characterised. Here, we report six individuals, from three independent families, harbouring biallelic variants in PTPMT1 with a complex neurological and neurodevelopmental syndrome. Using patient-derived fibroblasts and skeletal muscle tissue, together with cellular rescue experiments, we characterise the molecular defects associated with mutant PTPMT1 and confirm the downstream pathogenic effects that loss of PTPMT1 function has on mitochondrial structure and function. To further characterise the functional role of PTPMT1 in CL homeostasis, we established a zebrafish ptpmt1 knockout model associated with abnormalities in body size, developmental alterations, decreased total CL levels, and oxidative phosphorylation deficiency. Together, these data indicate that loss of PTPMT1 function is associated with a new autosomal recessive PMD caused by impaired CL metabolism, emphasising the contribution of aberrant CL metabolism towards human disease and its importance in neurodevelopment. E-mail: m.falabella@ucl.ac.uk
Prof. Nicolo Tonali.Rational design approaches to interfere with protein misfolding and aggregation through peptidomimetic foldamers
April 12, 2024 2:00 pm
The aggregation of proteins into amyloid fibers is linked to more than 40 still incurable cellular and neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease and type 2 diabetes. The process of amyloid formation is a main feature in cell degeneration and disease pathogenesis. Many anti-amyloid molecules have been reported over the past 25 years and most of them belong to small molecules or antibodies. However, so far only one of the anti-amyloid drug candidates, Tafamidis, which inhibits transthyretin amyloidogenesis (TTR), and an antibody, Aducanumab, which targets aggregates of beta-amyloid peptide 1- 42 (Aβ1-42), reached the clinic. Peptides are an attractive alternative to small molecules and antibodies as anti-amyloid drugs, thanks to their improved efficacy, selectivity or specificity, and potency. However, very few of them have reached the (pre) clinical stages, and so far, none of them has reached the clinic. Peptidomimetic foldamers, bio-inspired by secondary structures of amyloid proteins, provide a promising alternative to peptides because they keep the specific side chains of a peptide sequence while having new and improved biological and pharmacokinetic properties and the possibility of adopting secondary structures frequently involved in protein- protein interactions. Rational design approaches to interfere with protein misfolding and aggregation through peptidomimetic foldamers (β-strand, PPII and α-helix) are here presented. The design has been based on both self-aggregation and cross- interaction processes. These foldamers have shown a good ability in vitro to interfere with the aggregation process of Aβ1-42 and tau, especially those with a stable secondary conformation. Circular dichroism analyses on the conformational change of the amyloid protein in the presence of these foldamers revealed their ability to stabilize intermediate conformations, which could be the reason for the reduced aggregation propensity and thus toxicity. These approaches represent a practical application of peptidomimetic foldamers in therapeutics, particularly in pathologies involving abnormal protein-protein interactions. 1. N. Tonali, L. Hericks, D.C. Schröder, O. Kracker, R. Krzemieniecki, J. Kaffy, V. Le Joncour, P. Laakkonen, A. Marion, S. Ongeri, V. I. Dodero, N. Sewald; ChemPlusChem 2021, 86(6), 840. 2. L. Ciccone, C. Shi, D. Di Lorenzo, A.C. Van Baelen, N. Tonali; Molecules 2020, 25(10), 2439. 3. Shi, C.; Kaffy, J.; Ha-Duong, T.; Gallard, J.-F.; Pruvost, A.; Mabondzo, A.; Ciccone, L.; Ongeri, S.; Tonali, N.; J. Med. Chem. 2023, 66 (17), 12005–12017.

2023


Prof. Massimiliano Aschi - Enzymes by the point of view of a computational chemist
June 1, 2023
In the last years the development of advanced algorithms as well as the increase of computer efficiency have allowed the extension of the tools of theoretical-computational chemistry for the study of complex systems such as biomacromolecules. In this respect several strategies, based on different aspects involved in such a fascinating topic, have been proposed in the last two decades and, presently, it is not possible to state what is the better tool for this purpose. The strategy developed in our laboratory, developed in the framework of quantum-chemistry and basic statistical mechanics will be qualitatively outlined in this seminar and some of the related applications, specifically devoted to better understand the essential features of enzyme activity, will be illustrated.
Prof. Giampietro Schiavo - The axonal transport machinery and its dysfunctions in neurodegenerative diseases
May 19 2023 - Online Seminar
The molecular mechanisms causing neuronal death in many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Charcot Marie Tooth (CMT) disease, are poorly understood. The key consequence of our incomplete understanding of disease pathogenesis is that there is a complete dearth of effective symptomatic treatments for these widespread global disorders, prompting the necessity for a step-change in treatment strategies to fight these pathologies. In this view, we are investigating ALS and CMT as disease paradigms to identify new, common targets for pharmacological intervention in these devastating pathologies. Recently, we uncovered alterations in axonal transport of several cytoplasmic organelles, such as mitochondria and signaling endosomes, at pre-symptomatic stages of ALS and CMT pathogenesis, suggesting that these impairments may play a causative role in disease onset and progression. Crucially, we have restored axonal transport to physiological levels at early symptomatic stages of disease, thus demonstrating that these pathological changes are fully reversible. In light of these results, our main goal is identifying novel signaling nodes that modulate axonal transport in healthy and diseased neurons. This will allow us to test the hypothesis that counteracting axonal transport deficits observed in neurodegenerative diseases, represents a novel, effective therapeutic strategy towards treating these pathologies.
Prof. Amnon Horovitz - The chaperonin GroEL nano-machine: allostery and function
May 12, 2023
Chaperonins are nano-machines that are built of two back-to-back stacked heptameric rings. They assist protein folding by undergoing large conformational changes that are controlled by ATP binding and hydrolysis. In the E. coli cell, only about 60 different proteins require GroEL for efficient folding. In the first part of the talk, I will describe work that was aimed at determining the properties that distinguish GroEL clients from non-clients. In the second part of the talk, I will describe the impact of encapsulation on the stability of protein substrates. In the third part of the talk, I will describe new approaches for elucidating allosteric mechanisms. Using these approaches, it has been possible to show that GroEL undergoes concerted intra-ring conformational changes. By contrast, the eukaryotic homologue CCT/TRiC undergoes sequential intra-ring conformational changes. The impact of these different allosteric mechanisms on the folding functions of GroEL and CCT/TRiC will be discussed.
Prof. Lorenzo Stella - Exploring the allosteric mechanism of oncogenic phosphatase SHP2 
for the design of peptide inhibitors of its protein-protein interactions
May 5, 2023
The Src homology-2 domain-containing phosphatase 2 (SHP2), encoded by PTPN11, is a central node in RAS signaling and cancer and a pivotal target for anticancer therapy. PTPN11 mutations cause ~35% of cases of juvenile myelomonocytic leukemia (JMML) and occur in other malignancies. In addition, SHP2 is required for survival of RTK-driven cancer cells, contributes to resistance to anti-cancer drugs and modulates immune checkpoints. SHP2 comprises a catalytic PTP domain, and two SH2 domains (N-SH2 and C-SH2) mediating its interaction with phosphotyrosine-containing binding partners. Under basal conditions, the N-SH2 domain blocks the PTP active site and SHP2 is inactive. Most pathogenic mutations perturb this autoinhibited structure, enhancing basal phosphatase activity, simultaneously causing an increase in N-SH2 affinity for binding partners, through an allosteric mechanism. Several lines of evidence indicate that the latter effect is the main driver of RAS pathway hyperactivation caused by PTPN11 mutations. In this seminar I will illustrate our current structural and dynamical knowledge of the allosteric mechanism of SHP2 regulation. In addition, I will present a new class of inhibitors of SHP2 protein−protein interactions. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective on disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2−20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant in zebrafish embryos. Efforts to additionally improve the pharmacological properties of these inhibitors, to optimize their intracellular delivery and to explore other molecules with a similar mechanism of action are currently underway. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein−protein interactions in the function of SHP2.
Mirco Dindo - Regulation of enzymatic activity mediated by liquid-liquid phase separation
April 28, 2023
A feature of biological evolution at the cellular level is the development of diverse organelle structures in the cytoplasmic environment. Cells contain membrane-bound organelles and membrane-less organelles. Liquid–liquid phase separation (LLPS) is gaining acceptance as a powerful mechanism to explain the formation of membrane-less organelles. Emerging evidence indicates that LLPS is important in many biological processes, such as transcription, signaling and metabolism playing a vital role in human health and disease. These discoveries describe the LLPS as a new fundamental physicochemical mechanism for organizing the biochemistry of the cells. LLPS is recognized as a mechanism for regulation of enzymatic activity. Biochemical mechanisms include concentrating reactants to enhance reaction rates or sequester enzymes and reactants from each other to reduce the reaction rate. On the other hand, LLPS might also regulate the diffusion of small molecules or important parameters for enzymatic activity (such as modulators, macromolecular crowding and changing the media physicochemical features) increasing or decreasing the reaction rate of the enzymes. Here, I will show how LLPS contributed to generate a new era for enzymic activity regulation as well as other subtle regulation mechanisms still unexplored and recent advances in the LLPS field.
Prof. Simona Giunta - Protecting our genome: mechanisms to maintain DNA repeats stability in human cells
April 21, 2023
Centromere alpha-satellite DNA repeats are amongst the most recalcitrant regions of our genome. If we envisage genome browsers as Google Maps, zooming into increasing amount of details, centromeres have historically been black boxes that yield no linear sequence information. Yet, they are especially fascinating for three reasons: (1) centromeres are cytologically distinctive parts of human chromosomes that are essential for their segregation; (2) unlike the rest of our genome, centromere do not undergo meiotic recombination, hence they are inherited as intact blocks of maternal and paternal DNA; (3) in spite of points 1 and 2, centromere alpha-satellite DNA repeats are intrinsically unstable, a behavior of centromeres that I first described in 2018 as akin to human fragile sites in our genome. Indeed, centromere alpha-satellite repeats witness the convergence of several scenarios that can trigger DNA instability. Unlike the flanking region of constitutive heterochromatin at the pericentromere, centromeres are transcribed into non-coding RNAs throughout the cell cycle which offers opportunities for the collision between the transcription and replication machineries. Ours and others work have shown that centromere DNA is late replicating, prone to undergo rearrangements which becomes exacerbated in cancer and during cellular senescence. We demonstrated that epigenetic disruption of centromeric chromatin triggers pathological generation of mutagenic R-loops due to transcription-replication collision. The ensuing DNA damage repair within highly homogeneous centromere repeats results in a specific form of aneuploidy distinct from the numerical aneuploidy triggered by centromere inactivation, that we defined as structural aneuploidy including whole arms events and translocations frequently observed in most solid tumors. Our recent work shows that centromeres suffer higher level of endogenous DNA damage in unperturbed conditions compared to the rest of the genome, and also display different DNA damage repair kinetics, both contributing to their intrinsic instability. My laboratory uses genomics, including the latest human genome assemblies now including linear sequences of alpha-satellites, cytogenetics and high-end imaging to study centromeres as vulnerable regions of our genome. Altogether, work from my lab aims to understand centromere mutagenesis at the incipit of degenerative human diseases.
Dr. Velia Siciliano - Synthetic Biology: what, why and how
April 14, 2023
Synthetic Biology is a Bioengineering discipline that aims at reprogramming cell fate by designing genetic circuits that perform sophisticated information processing. Synthetic biology has the potential to revolutionize the treatment of hard-to-tackle diseases by reprogramming cells with synthetic devices. To obtain robust and specific activity, synthetic circuits must sense and respond to the intracellular or extracellular environment recognizing the unhealthy condition. Over the talk I will focus on the design of a platform that can be easily readapted to sense intracellular proteins of interest, and their application for engineering potential cell-based therapies. I will also show our work on RNA-encoded circuits that use RNA-binding proteins, siRNAs and proteases to engineer sensors, cascade and switches. Finally, I will present our recent research to address one of the standing bottlenecks of mammalian synthetic biology, namely the burden given by competition for intracellular resources that synthetic circuits impose to the cells.
Prof. Ping-Chih Ho - Educating tutor-associate macrophages under metabolic stress
March 30, 2023
Abstract. Metabolic stress is believed to impede proper activation and differentiation of immune cells in the tumour microenvironment. Moreover, re-educating tumour-associated macrophages is believed to be a critical process to reprogram immune state in tumors and maximise host anti-tumor immunity. In my talk, I will cover our understandings on metabolic regulations in macrophage polarisation and education. Then, I will further discuss how we can achieve efficient re-education in tumour-associated macrophages by requiring their metabolic programs.
Prof. Eugenio Barone - Insulin Signaling in the Brain and Models Thereof
March 23, 2023
Brain insulin signaling acts as a key regulator for gene expression and cellular metabolism, both events sustaining neuronal activity and synaptic plasticity mechanisms. Alterations of this pathway, known as brain insulin resistance, are associated with a higher risk to develop age-related cognitive decline and neurodegeneration.Among the molecular mechanisms identified to promote brain insulin resistance, mitochondrial dysfunctions, failure of energy metabolism, and increased oxidative stress levels have been found to play a role. Studies from our group uncovered the role of the enzyme biliverdin reductase A (BVRA) that, beyond its activity in the degradation pathway of heme, is a novel regulator of the insulin signaling. BVRA regulates insulin signaling pathway by working either as a S/T/Y kinase or a scaffold protein. In particular, BVRA is required to promote the AKT-mediated inhibition of GSK3β in response to insulin, that promotes cell metabolism and survival. Findings from our group revealed that a reduction of BVRA protein levels is a key event driving brain insulin resistance development. Moreover, we identified a novel mechanism for which loss of BVRA is responsible for GSK3β hyper-activation that drives mitochondrial stress and bioenergetics failure in response to insulin in neuronal cells. These alterations accelerate the impairment of energy metabolism and the development of neurodegeneration. Conversely, rescuing BVRA functions reduces oxidative stress levels, ameliorates brain insulin signaling activation and cellular metabolism, finally contributing to improved cognitive functions in animal models of neurodegeneration. Overall, our data suggest that BVRA links insulin signaling activation and mitochondrial bioenergetics to preserve cellular homeostasis and preventing the development of neurodegeneration.
Dr. Elena Enzo - Deciphering self-renewal traits in epidermal stem cells
March 17
Epithelial stem cells are endowed by impressive regeneration capacities, that sustain epithelia regeneration during homeostasis and wound healing in vivo. In vitro, they can proliferate for dozens of population doublings. Since the 1980s, cultured human keratinocytes have been extensively used to regenerate functional cornea and epidermis. Successful epithelia regenerations require an adequate number of epithelial stem cells, endowed by long lasting proliferative potential and self-renewing capacity. They progressively give rise to transient amplifying progenitors that proceed the onset of terminal differentiation. Despite their clinical success, we still are seeking a clear understanding of the molecular mechanisms that regulate self-renewal and regenerative potential self-renewal and regenerative potential in epithelial stem cells. Taking advantage of single-cell transcriptomic analysis of primary human epithelial cultures we identify the cluster of epithelial stem cells characterized by genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. We identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. Moreover, we are focusing on how stem cell and transient amplifying progenitors respond to genotoxic insults, looking at specifics molecular mechanisms that allow to prevent accumulation of genetic lesions in epidermal stem cells, and we found that FOXM1 is specifically activated upon genotoxic insults to foster DNA repair process. A better understanding of the complex network acting to repair the genotoxic insults will be instrumental to improve error-free based gene editing techniques. Moreover, as many genotoxic insults assault skin epithelia eventually causing oncogenic growth, this knowledge will also add insights on skin cancer progression, to develop early diagnostic tools or personalized therapies.
Prof. Anna Marabotti - Application of deep learning to the protein structure prediction: the tale of a “gigantic leap”
Thursday March 9, 2023
Knowing the three-dimensional structure of a protein is of outmost importance for understanding its function and evolutionary relationships with other members of the same protein family. However, the determination of 3D protein structures by experimental methods is not straightforward. For this reason, several computational methods have been developed in the past decades to predict the structure of proteins from their sequences. This field has experienced tremendous development in the last 4 years, when deep learning algorithms have made it possible to obtain structural predictions at a level of accuracy approaching that of experimental studies.
In this seminar, the evolution of protein structure prediction methods will be shown, with special reference to the development of artificial intelligence methods, and the implications of these new approaches for knowledge not only at the level of protein structure and function, but also in terms of knowledge of the interactions with other proteins and/or macromolecules inside and outside of cells, and, prospectively, for applying this knowledge in various fields, including rational drug design.
Prof. Cristian Ripoli - Engineering Proteins To Boost LTP And Memory
March 3, 2023
Synaptic plasticity (LTP) is considered the cellular correlate of learning and memory and its impairment has been linked to several neurological disorders, including Alzheimer's disease. Although the canonical pathways responsible for LTP involve dozens of proteins compartmentalized within dendritic spines, the LTP master controllers include key identified proteins such as CaMKII, LIMK1, PKC, BDNF and CREB. Using synthetic biology, we developed genetically encoded engineered proteins (GEEPs) to control their activity in living neurons boosting LTP. We employed artificial regulatory domains, whose conformations can be selectively and robustly controlled by the well-tolerated, clinically approved, blood-brain barrier permeant and procognitive drug rapamycin or its non-immunosuppressive analogs. Then, we combined biochemistry, electrophysiology, and two-photon imaging, demonstrating the viability of the proposed approach in controlling: •spine enlargement (GEE-LIMK1); •spine plasticity by either controlling BDNF maturation or engineering its receptor (GEE-BDNF/TrkB); •functional LTP (GEE-PKC) and •the clearance of misfolded proteins by engineering ADAM10. Using AAV infections and mouse behavioral analyses, we validated the potential of GEEPs to improve memory. Since several learning and memory disorders still lack efficacious treatments, our strategy will boost further research into next-generation bioengineering solutions as novel therapeutic strategies for in several brain disorders associated with cognitive dysfunctions.

2022


Dr Elizabeth Rhea - Regulation of insulin BBB transport: implications for Alzheimer’s
September 16
Dr. Elizabeth Rhea is a Research Assistant Professor at the University of Washington and Veterans Affairs Puget Sound in Seattle, WA. She has developed a great interest in the blood-brain barrier (BBB) and transport of key regulatory peptides, such as insulin, into the brain. While much is known about the impact of hormones within the CNS, little is known about how they are transported across the BBB. Therefore, she is further investigating transport properties and the role of BBB proteins and serum factors in BBB insulin transport. Her recent interests are on how the CNS can control the transport of insulin across the BBB. As CNS insulin resistance is a detrimental feature of Alzheimer’s disease, the regulation of insulin availability within the CNS could be an alternative therapeutic target.
Silvana Hrelia & Cristina Angeloni - Protective / Preventive Role Of Natural Compounds In Human Health: Biochemical Approaches To Target Ageing And Neurodegeneration
June 11, 2022
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. In this scenario, nutraceuticals constitute a unique source of privileged molecules with a safety profile and a vast multi-target potential.
Enrico Di Cera MD - Mechanisms of ligand binding
May 27, 2022
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.
Prof. Andrea Mattevi - NADPH Oxidase: Structure, enzymology and drug design
May 20, 2022
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, 10146
Alfredo Castello - When viral RNA met the cell: a story of protein-RNA interactions
May 13, 2022
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.
Michela A. Denti, PhD - RNA-based therapeutic approaches for neurodegenerative tauopathies
May 6, 2022
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.
Mauro Corrado - Metabolic control of T cell immunity
April 29, 2022
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.
Dr. Francesca Vallese - Architecture of the human erythrocyte ankyrin-1 complex
April 22, 2022
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 stoicheiometry of the heterotrimeric Rh channel, highlighting the role of ankyrin in mediating clustering of multiple copies of structurally diverse membrane proteins.
Alessandra Carattoli - Evolution of Klebsiella pneumoniae high-risk clones to pan-resistance
April 8, 2022
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.
Dr. Camilo Perez - Mechanism of a cell wall transporter involved in lipoteichoic acid synthesis and bacterial adaptation
April 1, 2022
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.
Alessio Paone - Amino acid metabolism: involvement in tumor progression and new therapeutic approaches
March 25, 2022
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.
Alfonso De Simone - Functional and Pathological Interactions of alpha-synuclein
March 18, 2022
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.
Alessandro Fatica “Emerging role for m6A RNA modification in cancer: learning from leukemia”
March 11, 2022
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.
Fundamentals of Enzyme Kinetics (3rd Edition)
February 24, 2022
The course is addressed to 1st & 2nd year students of the BeMM PhD School and is open to anybody who is interested.

2021


Prof. Dr. Stefan Knapp - Modulation of epigenetically controlled gene transcription and cell 
states by small molecules

July 19, 2021
Epigenetics is a key mechanism regulating context dependent gene expression and chromatin structure. One of the key questions in this area is how the epigenetic code is transferred during cell division leading to bookmarking of transcriptionally active genes and how is the code altered during cell differentiation and the development of diseases. We have been interested in the past 10 years in the reading process of the epigenetic code which is mediated by small protein interaction domains recognizing single or multiple post-translational modifications on histones and other nuclear proteins. One of the major epigenetic reader families are Bromodomains (BRDs), evolutionary conserved protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The human proteome encodes 61 of these highly diverse domains present in 46 mainly nuclear proteins. The recent discovery of potent and highly specific inhibitors for the BET (bromodomain and extra-terminal) family of bromodomains has stimulated intensive research activity in diverse therapeutic areas, particularly in oncology, where BET proteins regulate the expression of key oncogenes and anti-apoptotic proteins. During the recent years we have established a family wide platform of reagents, assays and crystal structures enabling the rational design and comprehensive selectivity screening of bromodomain inhibitors. Using this platform we and our collaborators have developed and comprehensively characterized highly selective chemical tool compounds (chemical probes) for most bromodomain subfamilies. In this talk I will present recent data on the developed tool compounds that cover now most bromodomain subfamilies including their in vitro characterization and phenotypic responses observed in cellular model systems as well as their potential for the development of new targeted therapies. I will also show examples how the unencumbered availability of chemical probes leads to rapid validation of new therapeutic strategies in diverse disease areas.
Prof. Dr. Stefan Knapp - The role of scaffolding and enymatic activity in kinase signalling. 
Lessons from studies with selective small molecule inhibitors.
July 16, 2021
In living cells, protein kinases are organized in large signalling complexes comprising adapter proteins, diverse enzymes and regulatory proteins. In recent years it has become increasingly evident, that protein kinases act not only as independent enzymes but that they also function as protein interaction scaffolds organizing the assembly of signalling complexes in a conformation sensitive way. This complexity is also reflected by the response of kinases to inhibitors that may stabilize diverse conformations acting as inhibitors of enzymatic activity only, as modulators of kinase scaffolding roles or both. In this talk, I will exemplify the implications of altering protein interactions by allosteric small molecules as well as canonical ATP competitive inhibitors using a number of selective inhibitors that we developed recently. I will demonstrate how different binding modes that alter protein conformation and dynamics in a distinct way may result in diverse signalling outcomes and phenotypic responses. The important scaffolding roles of protein kinases will also enable targeting new and so far poorly explored members of the kinase family such as catalytically inactive pseudokinases, that represent a considerable number of largely unexplored kinase targets which have been linked to the development of many diseases.
Dr. Gabriele Cerutti
Structural Basis for SARS-CoV-2 Neutralization by Human Antibodies
June 18, 2021
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.
Prof. Federico Bussolino
Transcription factor EB in vascular and heart biology
June 4, 2021
TFEB (Transcription factor EB) represents an emerging player inthe 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
Prof. Paolo Bernardi
Molecular nature and regulation of the mitochondrial permeability transition pore
May 28, 2021
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.
Dr. Philippa Mills
The evolving spectrum of vitamin B6 responsive disorders
May 21, 2021
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.
Prof. Jason LoCasale
Diet and Metabolic Therapeutics in Cancer
May 14, 2021
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.
Prof. Søren Riis Paludan
Identification of novel mechanisms in innate antiviral immunity
May 7, 2021
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.
Prof. Daniela Tropea
Rett Syndrome as a model to study neurobiology and candidate therapeutics for brain disorders
04/30/2021
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.
Dr. Tiziana Borsello
The Stressed Synapsis
23/04/2021
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.
Dr. Lovorka Stojic
Understanding how RNA-based mechanisms control genome stability in cancer
16 Aprile 2021
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.
Prof. Sarah Maria Fendt
Metabolic rewiring driving metastasis formation
April 9, 2021
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.
Dr Ina Huppertz, PhD
RNA regulates Glycolysis and Embryonic Stem Cell Differentiation via Enolase 1
March 26, 2021
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.
Prof Martino Bolognesi
Single particle cryo-electron microscopy, a quantum leap in structural biology
19/03/2021
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.
Prof. Romina Oliva
Analysis of protein-protein complexes and scoring of docking models
12/03/2021
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.
Dr Angelo Toto. Templated Folding of Intrinsically Disordered Proteins
05/03/2021
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.

2020


Live cell analysis of energy metabolism using the Hyp-ACB platform, a Sapienza research infrastructure at the Department of Biochemical Sciences - Prof. Serena Rinaldo
June 4 2020, 12 am online
Understanding how metabolic fluxes and gene expression can be turned on or off in a concerted manner is crucial to unravel the molecular mechanisms underlying physiological metabolic homeostasis and what goes awry in disease. Depicting possible metabolic re-programming also provide information on how biological systems react to environmental stimuli. Hypoxic Analysis of Cell Behaviour (Hyp-ACB) is a complex infrastructure able to measure, under normoxic and hypoxic conditions, the metabolic activity of living cells, with particular attention to the respiratory activity of mitochondria, in parallel with analysis of gene expression. The unique capability to monitor simultaneously cell metabolism and gene expression under variable oxygen tensions, allow to reproduce more closely the cell or tissue microenvironment(s) in both physiological and pathological conditions. Possible applications of this technology include multiple areas of biological research, spanning from molecular oncology, cardiovascular diseases, immunology, neurodegeneration, metabolic disorders, ageing, stem cells and tissue engineering, host-pathogen interactions, toxicology, drug discovery, environmental biology, bioengineering, food industry etc. Hyp-ACB is based on the scientific and technological multidisciplinary expertise already available in the Department, including redox biochemistry, kinetics of respiratory processes and cancer metabolism. An overview of the facility and selected examples of applications will be presented.
Che-1/AATF is involved in pediatric BCP-ALL onset and relapse: a new strategy to affect its expression and improve the current therapy - Dr. Valentina Folgiero PhD
May 29 2020, 12 am, online
Precursor B cell acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy and represents the leading cause of cancer-related death in children and young adults. BCP-ALL arises from a monoclonal or oligoclonal expansion of malignant B cell precursors in the bone marrow and despite the overall progress in treatment, relapse occurs across the whole spectrum of all subtypes with an overall survival of 30%. We recently found that Che-1/AATF, an important RNA polymerase II binding protein involved in the regulation of gene transcription with anti-apoptotic activity in different tumour contests, exerts a pivotal role in BCP-ALL. Che-1 is highly expressed in a cohort of BCP-ALL patients at the onset of disease and at time of relapse, irrespective of genetic heterogeneity, but was barely detectable when patients obtained leukemia remission. Notably, Che-1 depletion strongly affected growth of BCP-ALL cells and sensitized to Adriamycin. With the aim to therapeutically inhibit Che-1 expression, overcoming the difficulty of the nuclear localization, and to find a technical approach available in in vivo models we focused on exosome-mediated delivery of RNA oligos. This experimental approach resulted in downregulation of Che-1 expression in BCP-ALL cell lines. The obtained result produced also an increase in PARP cleavage and a higher amount of cells in subG0-1 phase. Since part of this procedure is an already approved GMP-grade procedure, we will test the cooperation between exosome-mediated Che-1 downregulation and the current therapeutic treatments with the aim to develop a new applicable clinical approach, particularly in BCP-ALL relapsed patients.
Mitochondria dynamism in T cells: of shape, movement, and cell physiology. Prof. Silvia Campello
May 22, 2020, 12 am, online
Mitochondria are dynamic organelles whose processes of fusion and fission are tightly regulated by specialized proteins, known as mitochondria-shaping proteins. Fusion and fission balance of the mitochondrial network is strictly required to regulate various processes, including the quality of mitochondria, cell metabolism, cell death, proliferation and cell migration. We study mitochondrial dynamics in lymphocytes physiology, and we recently highlighted their crucial role in several cell processes such as development, and proliferation, differentiation, migration, death. Interestingly, all these events are importantly involved also in T cell immune-surveillance and, in perspective, possible modulations of mitochondria morphology could open the way to future therapeutic approaches to modulate T cell response.
SARS-CoV-2 infection: the great fear and the urgent need of basic research - Prof Anna Teresa Palamara
May 19 2020 2 p.m. online
At the end of 2019 some cases of viral pneumonia emerged in the healthcare facilities of Wuhan, China. A novel strain of coronavirus was isolated on January 7th 2020 and sequenced on January 9th 2020 as a SARS-like virus. On February 2020 the WHO officially called the new virus SARS-Cov2 and named the disease COVID-19. The virus started to spread, initially as an epidemic. Subsequently, on March 11th 2020 the WHO declared the pandemic state. The virus affected millions of people causing thousands of deaths worldwide. The tremendous impact of SARS-CoV2 infection on health care systems, economy and everyday life, together with the lack of available vaccines or specific drugs, have shed light on the urgent need of scientific data to set up effective strategies able to fight the infection. In this context, the basic research plays a pivotal role in: -understanding the viral structure and the mechanisms of spread and transmissibility; -unraveling the complex relationship between virus and host that underpins the pathogenic mechanisms; -identifying novel potential targets for therapeutic and/or preventive strategies. In the seminar we will discuss the most recent findings on these aspects.
Architecture and Dynamics of Macromolecules Revealed by Cryo-Electron Microscopy Dr. Paolo Swuec
May 15 2020, 12 am, online
Over the last decade, cryo-electron microscopy (cryo-EM) has stepped up as the mainstream technology for studying the structure of cells, viruses and protein complexes at molecular resolution. In particular, single-particle electron microscopy has the ability to unravel the three-dimensional structure of biological molecules and assemblies by imaging non-crystalline specimens. The Cryo-EM Laboratory at University of Milan focuses on the architectural characterization of macromolecular complexes as targets for a deeper understanding of the molecular bases of both diseases and physiological pathways. In this talk, theoretical and practical aspects of single-particle EM will be addressed together with “real life” examples. In particular, the talk will cover the architectural characterization of (i) the anti-CRISPR protein AcrIIA6 from virulent streptococcal phage, (ii) the Azospirillum brasilense Glutamate Synthase in its different oligomeric assemblies, and (iii) cardiac amyloid fibrils from an immunoglobulin light chain (AL) amyloidosis patient.
Mitochondria in cancer: oncometabolites and beyond - Dr Christian Frezza
May 8, 2020, 12 am, online
The role of mitochondrial dysfunction in cancer has been debated for over a century. Recent bioinformatic data analyses revealed that mitochondrial genes are suppressed in cancer with poor clinical outcome. Furthermore, the fact that mutations of core metabolic enzymes in the mitochondria such as Fumarate Hydratase (FH) cause renal cancer strongly indicates that mitochondrial dysfunction can drive cancer. Today, I will provide an overview of our recent findings about the molecular mechanisms through which mitochondrial dysfunction can drive transformation and shape cancer progression, focusing on FH loss. Our work provides some insights into potential mechanisms of tumorigenesis caused by the accumulation of fumarate, a bona fide oncometabolite.
Nuclear transport receptors in cell division and cancer - Patrizia Lavia
April 24, 12 am, online
The function of proteins is dependent on their subcellular localization. Since the original discovery that proteins are “tagged” by signal sequences recognized by specific receptors that determine their localization, research efforts are relentlessly seeking to understand how these receptors work. Nuclear transport receptors, collectively called karyopherins, represent a large family, which include importins and exportins, of crucial importance in transporting proteins either to the nucleus or to the cytoplasm across the nuclear envelope in concert with the small GTPase RAN. In addition to this well-established function, work from our and other laboratories has shown that nuclear transport receptors take on new roles when cells enter mitosis and the nuclear envelope dissolves. Indeed: 1) classical experiments and “omics” approaches indicate that importin beta, the major nuclear import receptor, acts as a “master” regulator during mitosis and orchestrates the concerted localization of key factors at the mitotic apparatus and in chromosome segregation. 2) Importin beta is often deregulated in cancer and can be regarded as a proto-oncogene; this deregulated expression contributes to the genetic instability typical of cancer cells. 3) On the other hand, however, importin beta also modulates the sensitivity of cancer cells to therapeutic anti-mitotic drugs and thus represents a “vulnerability” that can be targeted in cancer. 4) Finally, an unexpected turn in importin function has emerged, with the recent rediscovery of an old inhibitor of parasitic infections, Invermectin, has proven capable to inhibit the replication of the sars-cov2 virus and has turned to act primarily as an inhibitor of importin beta. These data illustrate the multifaceted roles of nuclear transport receptors in cell life and duplication, with a wide reach in physiological processes and in diseases.
The Gain and the Loss function in the p53 business. Prof. Ivano Amelio
April 17, 2020, 12 am, online
The tumour suppressor protein p53, cooperated by its family members p63 and p73, has an essential role in the response to toxic injury. Somatic cells largely rely on p53 to overcome genotoxic stress and to maintain genomic integrity. Inactivation of p53 is indeed considered the “point of no return” for genomic instability. In addition to the canonical p53 control of cell cycle arrest/apoptosis, recent evidence indicates that upon cellular stress p53 coordinates highly diverse processes, such as cellular metabolism, redox homeostasis, and inter-cellular communication and interaction with the external micro‐environment. Hence, p53 is a critical factor in maintaining cellular homeostasis following a wide range of (micro)-environmental insults. I will discuss the contribution of p53 mutations to the cellular response to hypoxia, interactions with the cellular epigenome and the potential implications for acquisition of aggressive phenotype and genomic instability.
Vitamin B6 metabolism in health and disease. Prof. Roberto Contestabile
April 10, 2020, 12 am, online
The biologically active form of vitamin B6, pyridoxal 5’-phosphate (PLP), is a cofactor in over 200 enzyme activities involved in many metabolic pathways, including neurotransmitter synthesis and degradation. In humans, PLP is recycled from food and from degraded PLP-dependent enzymes in a salvage pathway requiring the action of pyridoxal kinase, pyridoxine 5’-phosphate oxidase and phosphatases. Once PLP is made, it is targeted to the dozens of different apoenzymes that need it as cofactor. The regulation of the salvage pathway and the mechanism of targeting of PLP to the apoenzymes are poorly understood. Severe neurological disorders, such as convulsions, epileptic encephalopathy and axonal polyneuropathy, result from inborn errors in proteins involved in vitamin B6 metabolism. This seminar will present the latest achievements in this field, focusing on the molecular basis of the above-mentioned neurological diseases and on the mechanisms of PLP homeostasis.
Order-disorder transition and conformational equilibria of PLP dependent enzymes. Dr. Giorgio Giardina
April, 3d 2020, 12pm online
Pyridoxal phosphate dependent enzymes are key metabolic enzymes whose dysfunction causes a plethora of neurological disorders and disease. On the other hand, many PLP dependent enzymes are upregulated in highly proliferating cells because of their metabolic reprogramming, and therefore they are good targets for anti-cancer therapies. The catalytic mechanism of PLP enzymes has been long studied and is well established. However, in recent years it has become increasingly clear that these enzymes have multiple roles and participate in the regulation of many different cell processes though an interplay of protein-protein and protein-nucleic acid interactions. These interactions are often induced by an order-disorder or conformational transition of the enzymes. The seminar will review the structural results that have contributed to change our perspective on the complex conformational landscape of three PLP dependent enzymes and their regulatory mechanisms.

2019


Selective Targeting of Kinase Catalytic and Non-Catalytic Function - Prof. Stefan Knapp
31 maggio
Protein kinases are enzymes with remarkable domain plasticity that plays a key role not only in their regulation but also in the mode of action of inhibitors and drugs targeting these dynamic systems. In this presentation I will outline some of the challenges targeting conformational states of kinases as well as new opportunities for drug discovery. I will present structure based design strategies for the development of highly selective inhibitors and consequences targeting different activation states of kinases interfering with scaffolding function. In addition, the importance of target residency, structural mechanisms leading to slow off-rates and methods measuring these parameters in cellular systems will be discussed. References: - Chaikuad, A., E, M.C.T., Zimmer, J., Liang, Y., Gray, N.S., Tarsounas, M., and Knapp, S. (2014). A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics. Nature Chemical Biology 10, 853-860. - Muller, S., Chaikuad, A., Gray, N.S., and Knapp, S. (2015). The ins and outs of selective kinase inhibitor development. Nature chemical biology 11, 818-821. - Chaikuad, A., Koch, P., Laufer, S.A., and Knapp, S. (2018). The Cysteinome of Protein Kinases as a Target in Drug Development. Angew Chem Int Ed Engl 57, 4372-4385. - Georgi, V., Schiele, F., Berger, B.T., Steffen, A., Marin Zapata, P.A., Briem, H., Menz, S., Preusse, C., Vasta, J.D., Robers, M.B., et al. (2018). Binding Kinetics Survey of the Drugged Kinome. J Am Chem Soc 140, 15774-15782. - Heroven, C., Georgi, V., Ganotra, G.K., Brennan, P., Wolfreys, F., Wade, R.C., Fernandez-Montalvan, A.E., Chaikuad, A., and Knapp, S. (2018). Halogen-Aromatic pi Interactions Modulate Inhibitor Residence Times. Angew Chem Int Ed Engl 57, 7220-7224. - Vasta, J.D., Corona, C.R., Wilkinson, J., Zimprich, C.A., Hartnett, J.R., Ingold, M.R., Zimmerman, K., Machleidt, T., Kirkland, T.A., Huwiler, K.G., et al. (2018). Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell Chem Biol 25, 206-214 e211.
Rock and Switch. The Mechanism of Transport of Solutes Across the Membrane by the Major Facilitator Superfamily Members - Prof. Fabio Polticelli
24 maggio
Members of the "Major Facilitator Superfamily" of transporters are ubiquitous membrane proteins responsible for transporting substrates such as metal ions, metabolites and small peptides. The transport mechanism includes three main conformational states, an inward open state, an occluded state and an outward open state. Depending on the direction of transport, these proteins bind their substrate in an open state and, passing through the occluded state, release it on the opposite side of the membrane. This mechanism is called "rocker-switch movement" and is regulated by the formation and rupture of precise interaction networks between amino acids, networks that are widely conserved in the family. These principles will be illustrated in detail using as a model ferroportin, the transporter involved in the iron efflux from eukaryotic cells, and its only known bacterial homolog, recently identified in the bacterium Bdellovibrio bacteriovorus.
The Eye as a Window on the Brain: Plaques, Tangles and Glial Activation as Retinal Biomarkers for Alzheimer's Disease - Prof. Silvia Diangeloantonio
17 maggio
Alzheimer’s disease is the most common cause of dementia and one of the leading sources of morbidity and mortality in the aging population. The brain AD pathology is characterized the accumulation of extracellular amyloid-beta peptides, derived from the cleavage of amyloid precursor protein, intracellular deposits of hyper-phosphorylated tau, neurodegeneration, and glial activation. However neuronal and glial modifications occur in the brain long before cognitive deficits, and clinical trials failed, maybe also because of the lack of an early diagnosis. The actual challenge is to define new biomarkers and non-invasive technologies to measure neuropathological changes in vivo at pre-symptomatic stages. Recent evidences on human samples and mouse models indicate the possibility to detect protein aggregates and other hallmarks in the retina, paving the road for non-invasive rapid detection of Alzheimer’s disease biomarkers. Here we demonstrate the presence of known and new retinal biomarkers in human retina of Alzheimer’s disease patients. We found the presence of amyloid beta plaques, tau tangles, neurodegeneration and astrogliosis in the retinal ganglion cell layer. Moreover, retinal microglia showed a disease associated microglial phenotype. We hypothesize retina as a window through which monitor Alzheimer’s disease -related neurodegeneration process.
The Emerging γ-Secretase Interactome and its Implication in the Pathogenesis oif Alzheimer’s Disease - Prof. Patrick Fraering
10 maggio
γ-secretase is an aspartyl protease that controls regulated intramembrane proteolysis of a growing list of single-pass type-I transmembrane proteins, including the amyloid precursor protein (APP) and the Notch-1 receptor. Importantly, γ-secretase is responsible for the final step in the production of amyloid-β peptides (Aβ), the key causative agents of Alzheimer’s disease (AD). Because an age-dependent dysregulation of its specific activity has pointed to the sporadic forms of AD, it remains critical to identify γ-secretase modulators (i) to better understand the biological mechanisms that cause AD, and (ii) for the development of therapies to safely treat this neurodegenerative disorder. We investigated the γ-secretase interactome and identified new endogenous modulators of APP processing and Aβ production. Among these, some play an important role in spatial learning and memory, while changes in their gene expression profiles were found in human brains from neuropathologically-verified AD cases. Keywords: γ-secretase, intramembrane proteolysis, interactome,
 Alzheimer’s disease, learning and memory.
Issues Involved in Patenting Biotechnology Inventions - Dr. Valentina Predazzi
3 maggio
Should your research be protected? Can your research be protected? Despite the critical role that biotechnology plays in saving, improving, and extending human life, there is a complicated process behind the work of patenting the inventions in this technical field. Researchers are often misinformed about the role and the possibilities arising around patents, so the main aim of the seminar will be to explain what patents really are, how they work and their importance in the field of biotechnology. By way of the example general guidelines will be presented on the types of patent protection available for inventions arising from research in the field of monoclonal antibodies, using concepts drawn from European case law and expert practice. Finally it will be briefly presented the patent battle between two educational institutions for the rights on CRISPR–Cas9 gene editing technology.
Effects of Proteasome Modulation in Ageing and Age-Related Diseases -Prof. Niki Chondrogianni 

18 Aprile
Proteasomes are constituents of the cellular proteolytic networks that maintain protein homeostasis through regulated proteolysis of normal and abnormal (in any way) proteins. Proteasome activation in cell lines has been shown to result to cellular lifespan extension and to exert protein anti-aggregation activity. Using Caenorhabditis elegans as a model, we analyzed in detail the proteasome status upon the progression of ageing and Alzheimer's disease (AD) and we investigated the effects of enhanced proteasome activities on the progression of the above mentioned phenomena. The obtained results were validated in human and murine cells of neuronal origin. More specifically, proteasome activation in C. elegans either through genetic means or through compounds resulted in enhanced levels of proteasome activities that led to a SKN-1- and proteasome activation-dependent lifespan extension. The elevated proteasome function conferred lower paralysis rates in various AD nematode models accompanied by decreased Aβ deposits thus ultimately decelerating the progression of AD phenotype. More importantly, similar positive results were also delivered in human neuroblastoma cells and in murine cortical neurons. Based on these results we have searched for natural and synthetic compounds with proteasome activating properties and we have found lead compounds that are currently under detailed investigation. Preliminary results revealed their beneficial effects against ageing. In total, our results suggest that proteasome activation with downstream positive outcomes on ageing and AD, an aggregation-related disease, is feasible in both a genetic and a non-genetic manipulation manner in cells as well as in a multicellular organism. Moreover they unveil the need for identification of anti-ageing and anti-amyloidogenic compounds either through chemical synthesis or among the nutrients found in our normal diet.
Unravelling Antimicrobial Peptides for Treatment of Pseudomonas Infections: Efficacy Studies and Nanotechnology Approaches for their Delivery - Prof. Maria Luisa Mangoni
12 aprile
Pseudomonas aeruginosa is among antibiotic-resistant bacterial pathogens for which new drugs are needed. It causes a large variety of infections including pneumonia, especially in cystic fibrosis sufferers, and keratitis in contact lens wearers. We discovered that the antimicrobial peptide (AMP) Esc(1-21) rapidly kills this pathogen limiting the induction of resistance. Furthermore, two selective L-to D-amino acid substitutions were found to make this peptide less cytotoxic, more stable and with a better in vivo efficacy in a mouse model of Pseudomonas-induced pneumonia or keratitis. One of the drawbacks in developing AMPs as new therapeutics is their inefficient delivery to the target site. We discovered that polymeric nanoparticles made of poly(lactic-co-glycolic) acid represent a promising tool for pulmonary delivery of peptides and their controlled release. We also found out that immobilization of these peptides to soft contact lenses is an effective strategy to achieve an antimicrobial medical device to prevent bacterial adhesion. Finally, we showed how conjugation of Esc(1-21) to gold-nanoparticles significantly increases its microbicidal activity without harmful effects to mammalian cells.
Human Neural Stem Cells as a Model to Study Neurodegenerative and Neurodevelopmental Diseases - Dr. Jessica Rosati
5 aprile
Today, the production of induced pluripotent stem cells (iPS) is an excellent method for creating human model systems for studying rare genetic diseases, about which little is yet known.  The subsequent differentiation into any kind of cell, and in particular, into cells from the nervous system, means that this model can be extended to include neurodegenerative and neurodevelopmental diseases in general, both hereditary and non-hereditary.​ 
The structure-toxicity relationship of various forms of alpha-synuclein associated with Parkinson’s disease - Prof. Fabrizio Chiti
29 maggio
Parkinson’s disease is associated with the conversion of the soluble protein -synuclein into protein aggregates (Lewy bodies) accumulating in the cytosol of dopaminergic neurons of the brain. We have isolated five species of the protein including the monomer (M), nontoxic type A* oligomers (OA*), toxic type B* oligomers (OB*), short (SF) and long (LF) fibrils. By using solution and solid-state NMR techniques and other biophysical techniques, we have identified the fundamental structural characteristics that enable toxic OB* oligomers, unlike OA*, to perturb biological membranes and generate cellular toxicity. We have then compared the evolution in time of six distinct readouts of cellular dysfunction in neuroblastoma cells and primary neurons, identifying the most toxic species and the time scale of the various events in the toxicity process. SF and LF induced the same cascade of toxic events as OB*, but slowly due to their ability to release slowly small oligomers close to OB*, which were observed to interact and cross neuronal membranes. We will finally show the ability of a small molecule called squalamine, which is currently under a phase II clinical trial to cure Parkinson’s disease, to inhibit the membrane-induced aggregation of alpha-synuclein and prevent the OB* interaction with biological membranes.
Alterazioni del metabolismo energetico cerebrale nel diabete insulino-resistente - Prof. Joao Duarte
15 marzo
The increasing prevalence of type 2 diabetes (T2D) in western societies is closely associated with obesity, sedentary life-styles and the excessive consumption of food products rich in fat and sugar. The whole-body metabolic imbalance in diabetes has a detrimental impact on brain function, leading to increased risk of dementia. Our lab focuses on understanding early metabolic dysfunction that might precede and be involved in the diabetes-induced neurodegenerative process. This lecture will cover our recent research on animal models of T2D, revealing alterations of energy metabolism in neurons and astrocytes caused by insulin resistance. It will also be discussed whether the loss of metabolic regulation in these brain cells can lead to synaptic dysfunction and memory impairment. Furthermore, I share recent experiments on neuroprotective strategies that can rescue brain function in T2D.
Protein dissection approach: a powerful tool 
in drug discovery processes - Prof. Daniela Marasco
22 marzo
Protein three dimensional structure is the complex recapitulation of local and distant intramolecular forces that cooperatively contribute to maintain finely tuned energetic equilibria. Secondary structure motifs and small domains might act as building blocks whose characterization would gain insights into the protein global structure but also to modulate interactions with external partners in its interactome [1]. Furthermore normally folded proteins can access to amyloidogenic states that are often considered as an ensemble of native-like conformations with locally unfolded elements. The characterization of intermediate amyloidogenic species is crucial to elucidate potential aggregation under native conditions and for in vivo aggregation events [2]. The talk will be focused on interdisciplinary approaches for the investigations of protein structure, function and evolution. Examples of protein dissection investigations will be reported both for the identification of potential new drugs in inflammatory/cancer diseases [3] and to investigate the destabilisation, aggregation, toxicity and cellular mislocalisation of nucleolar proteins [4] to explore new therapeutic ways. References [1] M.R. Hoopmann, R.L. Moritz, Current opinion in biotechnology, 24 (2013) 31-38. [2] F. Chiti, C.M. Dobson, Annual review of biochemistry, 86 (2017) 27-68 [3] S. La Manna, E. Lee, M. Ouzounova, C. Di Natale, E. Novellino, A. Merlino, H. Korkaya, D. Marasco, International journal of cancer, (2018). [4] P.L. Scognamiglio, C. Di Natale, M. Leone, R. Cascella, C. Cecchi, L. Lirussi, G. Antoniali, D. Riccardi, G. Morelli, G. Tell, F. Chiti, D. Marasco, Oncotarget, 7 (2016) 59129-59143.
Metabolic Paths to Neurodegeneration: Biochemistry holds the Key - Prof. Marzia Perluigi
8 marzo 2019
In recent years, it has become evident that metabolic alterations strongly influence the instigation and progression of many neurodegenerative disorders. Decreases in the functionality of several energy metabolism-related pathways in brain cells including glucose transport, mitochondrial electron transport, DNA repair, and neurotrophic factor signaling occur during normal aging and are further exacerbated in disorders such as Alzheimer's (AD), amyotrophic lateral sclerosis (ALS), Parkinson's (PD), and Huntington's (HD) diseases. Detailed knowledge of biochemical mechanisms that regulate cellular metabolism and signaling is central to understand how metabolic defects translate into a pathological phenotype.

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