Il Nobel per la medicina 2016 alle scoperte sulla "pulizia cellulare" Il Nobel per la medicina 2016 è stato assegnato a Yoshinori Ohsumi per le sue scoperte sui meccanismi dell'autofagia, un processo fondamentale di "pulizia cellulare", che ha un ruolo importante in molte patologie, da quelle neurodegenerative a malattie da accumulo lisosomiale a varie forme di tumore

‘autophagy’, =‘eating of self’ The word autophagy is derived from Greek words “auto” meaning self and “phagy” meaning eating: ‘autophagy’, =‘eating of self’ The term was first coined by Christian de Duve over 40 years ago, and was largely based on the observed degradation of mitochondria and other intra-cellular structures within lysosomes of rat liver perfused with the pancreatic hormone, glucagon.

In recent years the scientific world has ‘rediscovered’ autophagy, with major contributions to our molecular understanding and appreciation of the physiological significance of this process coming from numerous laboratories. Autophagy is a normal physiological process in the body that deals with destruction of cells in the body. It maintains homeostasis or normal functioning by protein degradation and turnover of the destroyed cell organelles for new cell formation. During cellular stress the process of Autophagy is upscaled and increased. Cellular stress is caused when there is deprivation of nutrients and/or growth factors. Thus Autophagy may provide an alternate source of intracellular building blocks and substrates that may generate energy to enable continuous cell survival. Currently, 32 different autophagy-related genes (Atg) have been identified by genetic screening in yeast and, significantly, many of these genes are conserved in slime mould, plants, worms, flies and mammals, emphasizing the importance of the autophagic process in responses to starvation across phylogeny . Although the importance of autophagy is well recognized in mammalian systems, many of the mechanistic breakthroughs in delineating how autophagy is regulated and executed at the molecular level have been made in yeast (Saccharomyces cerevisiae).

Figure 1. Autophagy regulatory pathway Figure 1. Autophagy regulatory pathway. After autophagy induction, the Atg1 complex (Atg1–Atg13–Atg17–Atg29–Atg31) translocates to the endoplasmic reticulum (ER), which is thought to be the major membrane source for autophagy (other membrane sources may include mitochondria and the plasma membrane). This leads to recruitment of the autophagy-specific form of the phosphatidylinositol 3-kinase (PI(3)K) complex, which includes Vps34, Vps15, Atg6/Beclin-1 and Atg14, to the ER. To form an autophagosome, elongation and closure of the isolation membrane requires 2 protein conjugation systems, the Atg12–Atg5–Atg16 complex and the Atg8/LC3–phosphatidylethanolamine (PE) complex. See text for more details. Published in: Lin Lin; Eric H Baehrecke; Molecular & Cellular Oncology  2015, 2, DOI: 10.4161/23723556.2014.985913 Copyright © 2015 The Author(s)

The molecular biology of autophagy Fougeray, S. & Pallet, N. (2014) Mechanisms and biological functions of autophagy in diseased and ageing kidneys Nat. Rev. Nephrol. doi:10.1038/nrneph.2014.201 Activation of AMPK in response to low cellular energy status inhibits mTOR and activates ULK1 via phosphorylation. Coordinated activation of the ULK1–ATG13–ATG101–FIP200 complex (which is inhibited by mTORC1) and the lipid kinase Vps34–Beclin 1 complex (which is inactivated by Bcl-2), drives the formation of the isolation membrane. The transmembrane proteins ATG9 and VMP1 participate in the recruitment of lipids from endosomes and the Golgi to the isolation membrane. The ATG5–ATG12–ATG16L1 complex and LC3–PE system also have roles in cargo recruitment, membrane elongation and maturation. ATG7 and ATG10 catalyse the conjugation of ATG12 to ATG5, whereas ATG7 and ATG3 catalyse conjugation of PE to LC3. The ATG5–ATG12–ATG16L1 complex covalently links PE to LC3, which becomes lipidated and associates with autophagosomes. SNARE proteins mediate fusion between autophagosomes and lysosomes and various lysosomal enzymes hydrolyze proteins, lipids and nucleic acids. Abbreviations: AMPK, AMP-activated protein kinase; ATG, autophagy-related protein; ATG16L1, Autophagy-related protein 16-1; DEPTOR, DEP domain-containing mTOR-interacting protein; FIP200, FAK family kinase-interacting protein of 200 kDa; LC3, microtubule-associated protein 1 light chain 3; mLST8, target of rapamycin complex subunit LST8; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; PE, phosphatidyl ethanolamine; PRAS40, proline-rich AKT1 substrate 1; Raptor, regulatory-associated protein of mTOR; SNARE, soluble NSF attachment protein receptor; ULK1, serine/threonine-protein kinase ULK1; VMP1, vacuole membrane protein 1; Vps, vacuolar protein sorting.

Autophagy LRRK2

Autophagy and cell death Autophagy also kills the cells under certain conditions. These are form of programmed cell death (PCD) and are called autophagic cell death. Programmed cell death is commonly termed apoptosis. Autophagy is termed a non-apoptotic programmed cell death with different pathways and mediators from apoptosis. Autophagy mainly maintains a balance between manufacture of cellular components and break down of damaged or unnecessary organelles and other cellular constituents.

Autophagy and stress Autophagy enables cells to survive stress from the external environment like nutrient deprivation and also allows them to withstand internal stresses like accumulation of damaged organelles and pathogen or infective organism invasion. Autophagy is seen in all eukaryotic systems including fungi, plants, slime mold, nematodes, fruit flies and insects, rodents (laboratory mice and rats), humans.

Types of autophagy There are several types of Autophagy. These are:- microautophagy – in this process the cytosolic components are directly taken up by the lysosome itself through the lysosomal membrane. macroautophagy – this involves delivery of cytoplasmic cargo to the lysosome through the intermediary of a double membrane-bound vesicle. This is called an autophagosome that fuses with the lysosome to form an autolysosome. Chaperone-mediated autophagy – in this process the targeted proteins are translocated across the lysosomal membrane in a complex with chaperone proteins (such as Hsc-70).   micro- and macropexophagy piecemeal microautophagy of the nucleus cytoplasm-to-vacuole targeting (Cvt) pathway

Nature LETTER doi:10.1038/nature14506 Receptor-mediated selective autophagy degrades the endoplasmic reticulum and the nucleus Keisuke Mochida1, Yu Oikawa2, Yayoi Kimura3, Hiromi Kirisako1,4, Hisashi Hirano3, Yoshinori Ohsumi2 & Hitoshi Nakatogawa1,4

ENDOCITOSI E’ UN MECCANISMO CON CUI LA CELLULA INTERNALIZZA ‘MATERIALI’ DALL’ESTERNO endosomi tardivi endosomi precoci Membrana TGN

VI SONO DIFFERENTI TIPI DI ENDOCITOSI TIPO DI MATERIALE INTERNALIZZATO FAGOCITOSI PINOCITOSI (micro-macro) TIPO DI VESCICOLA CHE SI FORMA ENDOCITOSI MEDIATA DA CAVEOLE (caveolina) ENDOCITOSI MEDIATA DA RECETTORE (clatrina)

batterio fagosoma endosoma precoce endosoma tardivo reticolo FAGOCITOSI endosoma precoce PINOCITOSI endosoma tardivo LISOSOMI reticolo endoplasmico mitocondrio autofagosoma AUTOFAGIA

TAPPE DI ENDOCITOSI MEMBRANA PLASMATICA ENDOSOMI PRECOCI MICROTUBULI CORPO MULTIVESCICOLARE ENDOSOMI TARDIVI TGN LISOSOMI

INDIRIZZI DELLA ENDOCITOSI

Compartimentalizzata Fosfatasi specifiche per PIP INOSITOLO, INOSITOLI FOSFATO ED IDENTITA’ DEI COMPARTIMENTI Gli inositolo fosfolipidi sono solo il 10% dei FL di membrana, ma hanno importanti funzioni di regolazione. Possono subire cicli di fosforilazione e defosforilazione dell’inositolo nelle posizione 3’,4’ e 5’ producendo tipi diversi di fosfoinositidi (PIP). La conversione di PI e PIP è altamente Compartimentalizzata Organelli e sia nella via biosintetica che secretoria contengono chinasi specifiche per PI e PIP Fosfatasi specifiche per PIP

DIVERSI TIPI DI FOSFATIDIL-INOSITOLO FOSFATO SONO GENERATI SULLA MEMBRANA Figure 13-10 Molecular Biology of the Cell (© Garland Science 2008)

DIVERSI TIPI DI FOSFATIDIL-INOSITOLO FOSFATO SONO PRESENTI SU DIFFERENTI DOMINI DI MEMBRANA Figure 13-11 Molecular Biology of the Cell (© Garland Science 2008)

LE PROTEINE Rab HANNO MOLTI INTERATTORI

Figure 13-15 Molecular Biology of the Cell (© Garland Science 2008)

FAGOCITOSI batterio pseudopodo membrana plasmatica

Figure 13-47b Molecular Biology of the Cell (© Garland Science 2008)

Motor Proteins and Exocytosis Power Phagocytosis From: Chavrier, Nature Cell Biol. 4:E169, 2002

Four major stages during phagosome formation. i) During ‘probing’, membrane extensions enriched in phagocytic receptors (blue) flail about in an actin-dependent manner, enhancing the likelihood of particle contact and receptor-ligand engagement. ii) During ‘early signaling and cup formation’, receptors cluster underneath the target particle, igniting a burst of tyrosine kinase activity that culminates in the recruitment of additional protein kinases, lipid kinases, adaptor proteins and GEFs that stimulate remodeling of the underlying actin skeleton. iii) During ‘pseudopod extension’, coordinated activation of lipid-modifying enzymes and an assortment of GTPases leads to a concerted assembly of highly dynamic actin filaments that drive the growth of membrane pseudopods to encircle the attached prey; localized secretion of endomembrane vesicles (yellow) provides extra area for membrane extension. iv) Lastly, during ‘phagosome closure’, the tips of the pseudopods meet and fuse, detaching the phagosome from the surface membrane. This is accompanied by signal abatement mediated by various lipid and protein phosphatases, as well as GAPs. The gray boxes provide a representative, but incomplete list of molecular regulators important for each stage of phagocytosis.

Figure I Phagocytosis of antibody (Ab)-coated particle. Trends in Microbiology 2013 21, 380-388DOI: (10.1016/j.tim.2013.06.001) Copyright © 2013 Elsevier Ltd Terms and Conditions

TAPPE DELLA ENDOCITOSI MEDIATA DA RECETTORE 1. INTERAZIONE LIGANDO-RECETTORE 2. LEGAME DELL’ADAPTINA AL RECETTORE 3. FORMAZIONE DEL COAT DI CLATRINA 4. INVAGINAZIONE DELLA MEMBRANA 5. FISSIONE 6. PERDITA DEL COAT DI CLATRINA 7. FUSIONE CON ENDOSOMI PRECOCI 8. ACIDIFICAZIONE 9. DISTACCO LIGANDO-RECETTORE 10. FORMAZIONE DI ENDOSOMI TARDIVI 11. TRASPORTO AI LISOSOMI E/O GOLGI (O RICICLO O TRANSCITOSI)

ENDOCITOSI MEDIATA DA CAVEOLINA E CLATRINA

IL RIVESTIMENTO DI CLATRINA HA UNA PRECISA ORGANIZZAZIONE GEOMETRICA

LA CLATRINA PROMUOVE LA FORMAZIONE DI VESCICOLE DI ENDOCITOSI ENDOCITOSI MEDIATA DA RECETTORE MOVIE

cargo recettore membrana CITOSOL adaptina Tf-R IL SEGNALE DI ENDOCITOSI VIENE RICONOSCIUTO DALLA ADAPTINA cargo recettore membrana CITOSOL adaptina Tf-R

ESEMPI DI ENDOCITOSI DI: 1 LDL 2 TRANSFERRINA 3 VIRUS 4 EGF 5 Ig POLIMERICHE

LE LDL TRASPORTANO COLESTEROLO 1 LE LDL TRASPORTANO COLESTEROLO Proteina Apo-B fosfolipidi colesterolo esteri del colesterolo

A coat di clatrina LDL recettori di LDL B recettori mutati

ENDOCITOSI DI LDL LDL recettori LDL endosoma enzimi colesterolo lisosoma

ENDOCITOSI DI LDL

A coat di clatrina LDL recettori di LDL B recettori mutati

2 LA TRANSFERRINA LEGA IL FERRO Fe Fe

ENDOCITOSI DELLA TRANSFERRINA recettori Fe Fe Fe Fe Fe Fe endosoma Fe Fe Fe Fe Fe Fe Fe Fe Fe Fe Fe transferrina enzimi lisosoma

4 L’ENDOCITOSI DELL’EGF SI REALIZZA TRAMITE IL SUO RECETTORE

IMPORTANZA DELLA UBIQUITINAZIONE Nell’endocitosi mediata da recettore I recettori vengono modificati dall’aggiunta di molecole di ubiquitina che servono a guidarli nel loro destino finale

The endosomal sorting complexes required for transport (ESCRT) machinery is made up of cytosolic protein complexes, known as ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Together with a number of accessory proteins, these ESCRT complexes enable a unique mode of membrane remodeling that results in membranes bending/budding away from the cytoplasm.[1][2] These ESCRT components have been isolated and studied in a number of organisms including yeast and humans.[3] The ESCRT machinery plays a vital role in a number of cellular processes including multivesicular body (MVB) biogenesis, cellular abscission, and viral budding. Multivesicular body (MVB) biogenesis is a process in which ubiquitin tagged proteins enter organelles called endosomes via the formation of vesicles. This process is essential for cells to destroy misfolded and damaged proteins.[4] Without ESCRT machinery, these proteins can build up and lead to neurodegenerative disease. For example, abnormalities in ESCRT-III components can lead to neurological disorders such as hereditary spastic paraplegia (HSP)

Figure 13-58 Molecular Biology of the Cell (© Garland Science 2008)

3 ENDOCITOSI DI VIRUS

Transcitosi delle IgG materne attraverso le cellule 5 Transcitosi delle IgG materne attraverso le cellule epiteliali dell’intestino di topi neonati

5 TRANSCITOSI

INGRESSO DI VIRUS IN UNA CELLULA

VIRUS DELL’INFLUENZA

SFV

IN CELLULE POLARIZZATE L’ESOCOTOSI DEL VIRUS E’ POLARIZZATA

Figure 13-61 Molecular Biology of the Cell (© Garland Science 2008)

membrana apicale endosoma apicale endosoma basolaterale membrana basolaterale

TRAFFICO VESCICOLE FUSIONE LIPIDI DI MEMBRANA VIRUS

TAPPE DI ENDOCITOSI MEMBRANA PLASMATICA ENDOSOMI PRECOCI MICROTUBULI TARDIVI TGN LISOSOMI

INDIRIZZI DELLA ENDOCITOSI RICICLAGGIO endosoma CITOSOL TRANSCITOSI DEGRADAZIONE lisosoma nucleo

LDL colesterolo fosfolipidi esteri del colesterolo proteina

Endocitosi di LDL

Figure 13-56 Molecular Biology of the Cell (© Garland Science 2008)

Figure 13-59 Molecular Biology of the Cell (© Garland Science 2008)