Learning objectives
Inform students of the main experimental strategies for large-scale studies of gene expression and protein function, and the profound consequences that these studies are having on our view of cells and organisms.
Prerequisites
Students should have a solid background in Chemistry, Biochemistry, Molecular Biology and recombinant DNA technologies.
Course unit content
<br />THE TERM PROTEOMICS INDICATES THE SYSTEMATIC IDENTIFICATION OF PROTEINS AND THEIR CHARACTERISATION WITH REGARD TO STRUCTURE, FUNCTION, ACTIVITY, QUANTITY AND MOLECULAR INTERACTIONS. THE PROTEOMIC APPROACH BEGAN TO PROVE TO BE EXTRAORDINARILY EFFECTIVE ONLY AFTER THE DETERMINATION OF THE COMPLETE SEQUENCE OF VARIOUS GENOMES, IN OTHER WORDS, IN THE POST-GENOMIC PHASE OF MODERN BIOLOGY. IDENTIFYING A PROTEIN BASICALLY MEANS KNOWING ITS PRIMARY STRUCTURE. UNTIL TEN YEARS AGO, THIS INFORMATION COULD BE OBTAINED ONLY WITH DIFFICULTY AND FOR VERY SIMPLE PROTEIN SAMPLES. CURRENTLY, IT CAN BE ACQUIRED EXTREMELY RAPIDLY, AND ALSO FOR HIGHLY COMPLEX PROTEIN MIXTURES. THE FACTORS THAT HAVE MADE THIS ADVANCE POSSIBLE ARE, ON THE ONE HAND, THE ALREADY MENTIONED AVAILABILITY OF COMPLETE GENOMIC DATABASES AND, ON THE OTHER, THE APPLICATION OF ADVANCED MASS SPECTROMETRY TECHNIQUES TO THE STUDY OF PROTEIN SAMPLES. THE SYSTEMATIC IDENTIFICATION OF ALL THE PROTEINS PRESENT IN A CELL LYSATE IS STILL AN ENORMOUS TASK AND, OF ITSELF, GIVES LITTLE INFORMATION. THIS IS WHY PROTEOMIC RESEARCH, ALTHOUGH HAVING VAST OBJECTIVES, GENERALLY STARTS FROM TARGETED QUESTIONS. FOR EXAMPLE: WHICH PROTEINS ARE OVER- OR UNDER-REPRESENTED IN A CELL POPULATION IN PATHOLOGICAL CONDITIONS? WHICH PROTEINS MAKE UP A PHYSICALLY ISOLATABLE SUB-CELLULAR PORTION THAT CARRIES OUT PRECISE FUNCTIONS (E.G. THE NUCLEOLUS, THE PROTEASOME, THE NUCLEAR PORE COMPLEX)? IN RECENT YEARS, PROTEOMIC STUDIES HAVE BEEN MADE PARTICULARLY INCISIVE BY THEIR UNION WITH PROTEIN LABELLING STRATEGIES, CALLED PROTEIN TAGGING. RELATIVELY SIMPLE PROCEDURES, BASED ON RECOMBINANT DNA TECHNOLOGY, ALLOW THE EXPRESSION OF PROTEIN VARIANTS CONTAINED IN A SHORT ADDITIONAL SECTION OF POLYPEPTIDE CHAIN (TAG) TO BE OBTAINED IN CELLS GROWN IN VITRO AND ALSO IN MULTICELLULAR ORGANISMS; THIS, FOR EXAMPLE, ALLOWS RAPID AND EFFECTIVE PURIFICATION OF THE LABELLED PROTEIN, OR ITS VISUALISATION IN VIVO, OR ITS PRECISE QUANTIFICATION. THE SYSTEMATIC LABELLING OF ALL THE PROTEINS OF A SIMPLE UNICELLULAR ORGANISM, THE SACCHAROMYCES CEREVISIAE YEAST, HAS ALLOWED THE COMPLETION OF STUDIES THAT WERE UNIMAGINABLE UNTIL A FEW YEARS AGO: THE SYSTEMATIC STUDY OF THE SUB-CELLULAR LOCALISATION OF ALL THE PROTEINS OF A CELL; THE STUDY OF THE INTERACTIONS OF EACH PROTEIN WITH ALL THE OTHER PROTEINS, AND THEREFORE THE CONSTRUCTION OF THE FIRST GLOBAL MAPS OF PROTEIN-PROTEIN INTERACTIONS; THE ABSOLUTE QUANTIFICATION OF THE LEVELS OF EACH PROTEIN IN THE CELL; THE IDENTIFICATION OF ALL THE REGIONS OF THE GENOME WITH WHICH A PARTICULAR NUCLEAR PROTEIN INTERACTS, AND THEREFORE THE CONSTRUCTION OF THE FIRST GLOBAL MAPS OF PROTEIN-DNA INTERACTIONS. SINCE ALMOST ALL PROTEINS ARE INVOLVED IN COVALENT MODIFICATIONS (E.G. PHOSPHORYLATION, GLYCOSYLATION, UBIQUITINATION) THAT REGULATE THEIR FUNCTION, RECENT YEARS HAVE SEEN THE HONING OF PROTEOMIC STRATEGIES THAT AIM AT SYSTEMATIC IDENTIFICATION OF ALL THE PROTEINS THAT, IN A CERTAIN CONDITION, BRING ABOUT A PARTICULAR COVALENT MODIFICATION. THE LONG-TERM, AND ALSO THE MOST AMBITIOUS, OBJECTIVE OF THE VARIOUS STRATEGIES MENTIONED IS THE CHANGE FROM A STATIC VIEW TO A DYNAMIC VIEW OF PROTEOMES. HOW DO CONCENTRATION, LOCALISATION, COVALENT MODIFICATIONS, MACROMOLECULAR INTERACTIONS AND THE ACTIVITY OF ALL THE PROTEINS OF A CELL CHANGE WHEN IT PASSES FROM ONE GROWTH CONDITION TO ANOTHER, OR FROM ONE PHASE OF ITS CYCLE TO ANOTHER, OR FROM A NORMAL TO A PATHOLOGICAL STATE?
Bibliography
During the course, teaching material consisting of reviews and original scientific articles in English, updated year-by-year, will be made available.
Teaching methods
The course is based on classroom lectures in which recent scientific articles of great impact are generally analysed in detail.
