Proteostasis and Therapeutic Development

ECTS

4

Learning outcomes

Protein misfolding is presently associated with a diverse and growing number of severe human pathologies (e.g. neurodegenerative diseases and specific cases of hereditary cancer) driving the development of new therapeutic approaches aimed at modulating the function of the misfolded target protein acting on its proteostasis. In this perspective, the CU aims to expand the knowledge about the molecular mechanisms underlying disturbed proteostasis and the experimental strategies to identify therapeutic targets and molecules with a potential therapeutic effect. In a theoretical, theoretical-practical and laboratory perspective, the students will acquire skills that will allow them to: Identify the intracellular protein quality control systems; Understand the pathogenic consequences of protein misfolding; Recognize if the proteins under study and the pathways of proteostasis are potential therapeutic targets; Be familiar with new technologies and model organisms to the study of pathways involved in the maintenance of a healthy proteome; Design strategies for the identification of molecules with therapeutic potential (pharmacological modulation of proteostasis); Learn how to manipulate scientific equipment and specialized software and how to analyze and present experimental data.

Syllabus

Theoretical course

Proteins: Structure and Function (Structural organization and stabilizing interactions; Post-translation modifications; Folding in vitro - thermodynamic and kinetic bases; folding models); Biosynthesis and Protein Processing (Folding in vitro/in vivo; The ribosome; Co- and post-translation folding; Folding in different cell compartments; The molecular chaperones and co-chaperones systems); Protein Degradation Pathways (Half-Lifetime; The N-degrons; The Ubiquitin-Proteasome and Autophagy-Lysosomal Systems; Degradation in the various cell compartments); Proteotoxic stress response systems (UPR and HSR); Conformational diseases (Loss-of-function and gain-of-function; Genetic diseases and missense mutations; Small molecules modulators of intracellular folding - pharmacological chaperones and  proteostasis regulators); Orphan Drugs (Rare Diseases; Orphan drugs Designation; Regulations and incentives; Committee of orphan drugs; Examples of orphan drugs).

Practical course

Protein expression and purification systems (Prokaryotic and eukaryotic systems; Peptides and fusion proteins; Co-expression systems; Purification methods); Laboratory tools for structural and functional protein characterization - In vitro methods (Non-Spectroscopic techniques; Spectroscopic techniques, Evaluation of biological activity; Binding Assays; Protein stability assays) and in cellulo methods (pulse-chase; Co-immunoprecipitation; BRET); Experimental approaches for the identification of pharmacological chaperones and proteostasis regulators (High-Throughput Screening Platforms; Compound Libraries; Compound screenings (random/oriented; in vitro/in cellulo); Bioinformatic tools for the study of proteins (Databases; Programs for comparative analysis of biological information; Bioinformatic tools for predicting changes in protein stability (DG); Programs for protein visualization)

Laboratory Course

Expression and purification of a eukaryotic protein, wild-type (WT) and a pathogenic variant (Transformation of competent cells; Preparation of Overnight; Cell Lysis; Purification of recombinant proteins by affinity chromatography and size exclusion; Determination of the yield of purified proteins); Functional characterization of recombinant proteins WT and pathogenic variant (Determination of enzymatic activity and kinetic parameters; Thermal inactivation assays); Structural characterization of recombinant proteins (Purity degree; Quaternary structure; Limited proteolysis (tertiary structure); Thermal stability); Identification of protein folding stabilizing molecules (DSF: Differential Scanning Fluorimetry).

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