Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B

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Thesis

Reference

Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B

CHIUSOLO, Valentina

Abstract

Granzyme B-induced apoptosis requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How granzyme B (GB), which does not possess a mitochondrial targeting sequence, enters this organelle is unknown. We show that GB enters mitochondria independently of the translocase of the outer mitochondrial membrane (TOM) complex but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane -barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat shock protein 70 (mtHsp70)-dependent manner. Granzyme A and caspase 3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated ROS and cell death. Therefore, cytotoxic molecules enter the mitochondria to efficiently induce cell death through a non-canonical Sam50, Tim22 and mtHsp70-dependent import pathway.

CHIUSOLO, Valentina. Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B. Thèse de doctorat : Univ. Genève, 2016, no. Sc. 4959

URN : urn:nbn:ch:unige-863877

DOI : 10.13097/archive-ouverte/unige:86387

Available at:

http://archive-ouverte.unige.ch/unige:86387

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE

Département de biologie cellulaire FACULTÉ DE SCIENCES Professeur Jean-Claude Martinou

Département de physiologie et métabolisme

FACULTÉ DE MÉDECINE Professeur Denis Martinvalet

Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Valentina CHIUSOLO de Benevento (Italie)

Thèse n° - 4959 -

Genève

Atelier de reprographie à Uni Mail

2016

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UNIVERSITÉ DE GENÈVE

Département de biologie cellulaire FACULTÉ DE SCIENCES Professeur Jean-Claude Martinou

Département de physiologie et métabolisme

FACULTÉ DE MÉDECINE Professeur Denis Martinvalet

Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Valentina CHIUSOLO de Benevento (Italie)

Thèse - 4959 -

Genève

Atelier de reprographie à Uni Mail

2016

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List of figures and tables

Figure 1: Hematopoietic stem cells. pag 9

Table 1 pag 12

Figure 2: Activation pathway of the complement system. pag 17

Figure3: CTL attacking a cancer cell. pag 21

Figure 4: Receptor-mediated cell death pathways. pag 23

Figure5: Perforin pore structure. pag 26

Figure 6: Gene clusters of human and mouse granzymes. pag 28

Figure 7: GB-mediated cell death pathways. pag 32

Figure 8: Mitochondrial import complexes. pag 46

Figures (Manuscript). pag 92-103

Supplementary figures (Manuscript). pag 104-115

Figure 9: Complex I subunits cleaved by GB, GA and caspase-3. pag 119 Figure 10: Schematic 3D-structure of human GA (A), mouse GB (B) and

human GM (M).

pag 120

Figure 11: CHCHD3, Mitofilin (MF) and Sam50 interactions. pag 123

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List of Abbreviations

3D: Three-dimensional Å: Angstrom

ADP: Adenosine diphosphate AICD: Activation-Induced Cell Death AIF: Apoptosis-Inducing Factor

Apaf-1: Apoptotic Protease Activating Factor-1 APC: Antigen-Presenting Cell

Ape-1: Apurinic Endonuclease-1 APO-1: Apoptosis antigen-1 Arg: Arginine

Asp: Aspartic acid or Aspartate

ATCC: American Type Culture Collection ATP: Adenosine triphosphate

Bak: Bcl-2 homologous antagonist killer BAM: β-barrel Assembly Machinery Bax: Bcl-2-associated X protein BCA: Bicinchoninic Acid Bcl-2: B-cell lymphoma-2 BER: Base Excision Repair

Bid: BH3-interacting domain death agonist BN-gel: Blue Native gel

BSA: Bovine Serum Albumin

C1, C2, C3, C4, C5, C6, C7, C8, C9: Complement component 1, 2, 3, 4, 5, 6, 7, 8, 9 C-terminus: Carboxyl terminus

Ca²⁺: Calcium

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Calcein-AM: Calcein-Acetoxymethyl CAM: Cell Adhesion Molecule Casp: Caspase

CD: Cluster of Differentiation

CDCs: Cholesterol Dependent Cytolysins cFLIP: Cellular FLICE-Inhibitory Protein CFSE: Carboxyfluorescein Succinimidyl Ester

CHCHD3: Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 3 CLR: C-type Lectin Receptors

CMA-1: Mast Cell-Chymase 1 coQ: Coenzyme Q

COX: Cytochrome c Oxidase

CpG: Cytosine-phosphate-Guanine deoxynucleotide motif CR: Complement Receptor

cSMAC: Central Supramolecular Activation Complex CTL: Cytotoxic T Lymphocyte

CuA, CuB: Copper atom A, B

CXCL8: C-X-C motif Chemokine Ligand 8 Cyt-c: Cytochrome c

DAF: Decay-Accelerating Factor DAPI: 4',6-diamidino-2-phenylindole DBP: DNA Binding Protein

DED: Dead Effector Domain DHE: Dihydroethidium

DHFR: Dihydrofolate Reductase

DIABLO: Direct Inhibitor of Apoptosis protein (IAP)-Binding protein with Low PI

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DISC: Death-Inducing Signaling Complex DKO: Double Knockout

DMEM: Dulbecco's Modified Eagle Medium DNA: Deoxyribonucleic acid

Dox: Doxicyclin

ΔpH: Delta potential of Hydrogen

Δψ: Mitochondrial transmembrane potential Drp1: Dynamin-related protein 1

DSP: Dithiobis (succinimidyl propionate) E:T: Effector:Target

ECM: Extracellular Matrix

EDTA: Ethylenediaminetetraacetic Acid EEA-1: Early Endosome Antigen-1 EGT: Endosymbiotic Gene Transfer EGTA: Ethylene Glycol Tetraacetic Acid EM: Electron Microscopy

ER: Endoplasmic reticulum

ERMES: Endoplasmic Reticulum-Mitochondria Encounter Structure ETC: Electron Transport Chain

FAD: Flavin Adenine Dinucleotide FADD: Fas-Associated Death Domain

Fas/FasL: First Apoptosis Signal/First Apoptosis Signal Ligand FBS: Fetal Bovine Serum

Fc: Fragment Crystallizable FCS: Fetal Calf Serum Fe-S: Iron-Sulfur

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FHL-2: Familial Hemophagocytic Lymphohistiocytosis type 2 FLICE: FADD-like IL-1β-converting enzyme

FMN: Flavin Mononucleotide FoxP3: Forkhead box P3

FRET: Fluorescence Resonance Energy Transfer GA: Granzyme A

GAGs: Glycosaminoglycans

GAPDH: Glyceraldehyde 3-Phosphate Dehydrogenase GB: Granzyme B

Gly: Glycine GM: Granzyme M GNLY: Granulysin

GTP: Guanosine Triphosphate G: Granzyme

Hax-1: HCLS1 (hematopoietic cell specific Lyn substrate 1)-associated protein X-1 HBSS: Hanks' Balanced Salt Solution

His: Histidine

HMGB2: High-Mobility Group Protein B2

hnRNP K: Heterogeneous nuclear Ribonucleoprotein K HRP: Horseradish Peroxidase

Hsp-27, -60, -70, -90: Heat-shock protein-27, -60, -70, -90 ICAD: Inhibitor of the Caspase-Activated DNase

ICAM-1: Intercellular Adhesion Molecule-1 IFNα, β, γ: Interferon α, β, γ

IL1R: Toll/interleukin-1-like receptor

IL-2, -6, -7, -12, -15 : Interleukin-2, -6, -7, -12, -15

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Ile: Isoleucine

ILT: Immunoglobulin-Like Transcript IMM: Inner Mitochondrial Membrane IMS: Intermembrane Space

IRF: Interferon Regulatory Factor IS: Immunological Synapse

ITS: Intermembrane space Targeting Signal kDa: Kilodalton

KIR: Killer-cell Immunoglobulin-like Receptor

Lamp-1, -2: Lysosome-Associated Membrane Protein-1, -2 LCMV: Lymphocytic Choriomeningitis Virus

LFA-1, -3: Lymphocyte Function-associated Antigen-1, -3 LILR: Leukocyte Immunoglobulin-Like Receptors

λPP: Lambda Protein Phosphatase LPS: Lipopolysaccharide

LRO: Lysosome-Related Organelle Lys: Lysine

MAC: Membrane Attack Complex

MACPF: Membrane Attack Complex Perforin domain MAPK: Mitogen-Activated Protein Kinase

MASP-1, -2: Mannan-binding lectin Serine Protease-1, -2 MBL: Mannose-Binding Lectin

MCP: Membrane Co-factor Protein

Mdm10: Mitochondrial distribution and morphology protein 10 Met: Methionine

mFAS: membrane-bound First Apoptosis Signal

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Mfn-1, -2: Mitofusin-1, -2

MHC-I, -II: Major Histocompatibility Complex-I, -II MIA: Mitochondrial Intermembrane Assembly machinery Mic19: MICOS complex subunit-19

MICOS: Mitochondrial Contact Site complex

MINOS: Mitochondrial Inner membrane Organizing System MOMP: Mitochondrial Outer Membrane Permeabilization MOPS: 3-(N-Morpholino) Propanesulfonic acid

Mort-1: Mediator Of Receptor-induced Toxicity-1 MPP: Mitochondrial Processing Peptidase

mt: Mitochondrial

MTOC: Microtubule Organizing Center MTX: Methotrexate

N1, N2: Neutrophils type 1 and type 2 N-terminus: Amino-terminus

NAD: Nicotinamide Adenine Dinucleotide

NADH: Nicotinamide Adenine Dinucleotide (reduced form) NADPH: Nicotinamide Adenine Dinucleotide Phosphate NDUFS-1, -2, -3: NADH Dehydrogenase Fe-S protein-1, -2, -3 NDUFV1: NADH Dehydrogenase [Ubiquinone]-Flavoprotein-1

NF-κB: Nuclear Factor kappa-light-chain-enhancer of activated B cells NK: Natural Killer

NKG2: Natural Killer Group 2 NLR: NOD-Like receptor NMII: Non muscle Myosin II

NM23-H1: Non-Metastatic protein 23-Homolog 1

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NOD: Nucleotide-Binding Oligomerization NPM: Nucleophosmin

NuMa: Nuclear Mitotic apparatus protein OH: Hydroxyl

Omi/Htra2: HtrA serine peptidase 2 OMM: Outer Mitochondrial Membrane Opa-1: Optic atrophy-1

Oxa-1, -2: Mitochondrial oxidase assembly protein-1, -2 OXPHOS: Oxydative phosphorylation

ρ°: Pseudo rho

PAM: Pre-sequence-Associated Motor

PAMP: Pathogen-Associated Molecular Pattern PARP-1: Poly (ADP-ribose)-Polymerase-1 PCR: Polymerase Chain Reaction

PFN: Perforin

pH: Potential of Hydrogen Pi: Inorganic phosphate PI-9: Protease inhibitor-9 PK: Proteinase K

Plu-MACPF: P.luminescens-Membrane Attack Complex/Perforin PMSF: Phenylmethylsulfonyl Fluoride

POTRA: Polypeptide-Transport-Associated domain PP2A: Protein Phosphatase 2A

pp32: Phosphoprotein 32

PRR: Pattern-Recognition Receptors PSTI: Pancreatic Secretory Trypsin Inhibitor

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PVDF: Polyvinylidene Difluoride Q: Ubiquinone

Rab5: Ras-related protein Rab5

RIG-1: Retinoic acid Inducible Gene-1 protein RIPA buffer: Radioimmunoprecipitation Assay buffer RNA: Ribonucleic Acid

ROS: Reactive Oxygen Species

RPMI: Roswell Park Memorial Institute medium rRNA: ribosomal RNA

RT: Room Temperature

RUNX3: Runt-related transcription factor-3

35S: Sulfur-35 isotope

SAM: Sorting and Assembly Machinery SAPLIP: Saposin-Like Protein

Sdh3: Succinate dehydrogenase cytochrome b subunit SDS: Sodium Dodecyl Sulphate

SEM: Scanning Electron Microscopy Ser: Serine

sFAS: soluble FAS

shRNA: short/small hairpin RNA

Smac: Second Mitochondria-derived Activator of Caspase SOD: Superoxide Dismutase

Ssc-1: Yeast mitochondrial Hsp70 TAN: Tumor-Associated Neutrophils tBid: Truncated Bid

TCA: Trichloroacetic acid

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TCRα, β: T-cell receptor α and β chain Th: Helper T cell

ThPOK: Th-inducing Pox virus and zinc finger/Krüppel-like factor TIM: Translocase of the Inner Membrane

TIR: Toll/Interleukin-1 Receptor TLR: Toll-Like Receptor

TNF: Tumor Necrosis Factor

TNFR: Tumor Necrosis Factor Receptor

TOB: Topogenesis of mitochondrial Outer-membrane Beta-barrel protein (yeast orthologs of mammalian SAM)

Toc75: Translocon of the Outer Chloroplast membrane-75 TOM: Translocase of the Outer Membrane

TopoIIα: DNA topoisomerase 2-alpha

TRAIL: Tumor Necrosis Factor-related Apoptosis-Inducing Ligand T_reg: Regulaotory T cell

TREX1: Three prime Repair Exonuclease-1 tRNA: transfer RNA

Trx: Thioredoxin Tyr: Tyrosine UV: Ultraviolet

XIAP: X-linked Inhibitor of Apoptosis Protein Val: Valine

VDAC: Voltage-Dependent Anion Channel vFLIP: viral FLICE-Inhibitory Protein Zn²⁺: Zinc

zVAD: Benzyloxycarbonyl-valine-alanine-aspartic acid

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Chapter I

RÉSUMÉ AND SUMMARY

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1.1. Résumé

Les lymphocytes T cytotoxiques (CTL) et les cellules NK (Natural Killer) permettent l’élimination des cellules malignes ou infectées par un virus. Leur capacité à tuer repose sur des protéines cytotoxiques contenues dans des granules cytoplasmiques spécialisées. Ces granules contiennent principalement la perforine et une famille de protéases à Sérine appelées granzymes, qui, lors de la formation d’un conjugué entre la cellule immunitaire et sa cible, sont rélarguées dans la cellule cible afin d’induire l’apoptose. Parmis les granzymes, granzyme A (GA) et granzyme B (GB) sont les plus abondants et les mieux caractérisés à la fois chez l’Homme et la Souris. GA, un homodimère de 50 kDa stabilisé par des ponts disulfures, déclenche une mort cellulaire indépendente des caspases en induisant une fragmentation rapide de l’ADN, une rupture de l’enveloppe nucléaire et une production massive de radicaux libres oxygénés (ROS). Notamment, GA clive et active le complexe SET associé au réticulum endoplasmique, ce qui permet la fragmentation de l’ADN. Sous l’effet du potentiel de membrane mitochondriale, GA est également capable d’entrer dans les mitochondries où il clive la sous-unité NDUFS3 du complexe I de la chaine respiratoire, induisant la production de ROS et la mort cellulaire. GB, une protéase de 32 kDa, est capable d’induire une mort cellulaire à la fois de manière dépendante et indépendante des caspases. Tout d’abord, GB est capable de cliver directement les caspases-3, -7, -8 et -10. GB partage également certains substrats avec les caspases, comme par exemple PARP-1, ICAD et la lamine B qui sont clivé après un résidu d’acide aspartique de la même manière que la caspase-3. Une voie mitochondriale de l’apoptose est également activée par GB de manière caspase-independante via le clivage de Bid qui conduit à l’oligomérisation de Bax et Bak et à la perméabilisation de la membrane externe mitochondriale (MOMP). Le relargage des facteurs apoptogéniques tels que le cytochrome c, AIF, Smac/DIABLO consécutivement au MOMP conduit à l’activation des caspases et à une amplification de la mort cellulaire. Tout comme GA, GB est également capable d’entrer dans la matrice mitochondriale sous l’action du potential de membrane, où il clive les sous-unités NDUFV1, NDUFS1 et NDUFS2 de la chaine respiratoire pour induire un défaut de la respiration mitochondriale et une production de ROS. L’action directe de GA et GB dans la mitochondrie suggère que ces 2 protéases doivent traverser de façon active à la fois les membranes internes et externes de la mitochondrie pour atteindre leurs substrats, par un mécanisme probablement partagé par tous les types de Granzymes. Or pour ce faire, ni GA, ni GB ne possèdent de peptide d’adressage mitochondrial connu. Nous nous sommes donc

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demandé comment ces protéases pouvaient atteindre la matrice mitochondriale. Mon travail de doctorat s’est focalisé sur la charactérisation du mécanisme moléculaire par lequel GB (mais aussi GA et la caspase-3) pénètre au coeur des mitochondries pour y cliver les sous- unités de la chaine respiratoire et y induire la mort cellulaire de manière ROS-dependante.

L’examen de la séquence peptidique, de la structure tridimensionnelle, ainsi que la réalisation de différents mutants des Granzymes nous a permis de comprendre que son hélice α C- terminale était responsable de l’import mitochondrial. Ce domaine dans la partie C-ter de GB est conservée chez tous les autres Granzymes et dans d’autres espèces et possède une série de résidus basiques rassemblés sur la face de l’hélice α exposée aux solvants. Il a été démontré auparavant que la charge positive de GB était importante pour son entrée dans le cytosol des cellules cibles lors de la formation de la synapse et la dégranulation. Nous montrons ici que les résidus basiques sont critiques pour l’import mitochondrial de GB et que leur absence entraîne une baisse du potentiel d’induction de la mort cellulaire in vitro et in vivo. L’absence d’une séquence d’adressage mitochondrial canonique suggère que l’import de GB ne suit pas la voie classique. En effet, nos experiences montrant sans équivoque que ce mécanisme est indépendent de la machinerie d’import TOM et TIM. En réalité, ni le canal Tom40, ni le canal Tim23, qui sont responsables de l’import de la quasi-totalité des proteins mitochondriales, ne sont necessaires à l’import de GB. Au contraire, nous avons montré que GA, GB et la caspase-3 traversent la membrane externe par le canal SAM50, tandis que la membrane interne est franchie via la protéine canal Tim22. De plus, nous avons montré que lorsque qu’un mutant de GB incapable d’atteindre la matrice mitochondriale était utilisé, la production de ROS et la mort cellulaire était réduites, ce qui supporte le concept inattendu que cette voie mitochondriale est cruciale dans le contexte de la mort induite par les CTL/NK. L’entrée des Granzymes par une voie non-conventionnelle montre qu’une autre entrée existe vers la mitochondrie et qu’elle pourrait aussi être utilisée par d’autres protéines pour accéder à cette organelle dans des contextes aussi bien physiologiques que pathologiques.

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1.2. Summary

Cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells target and kill virus-infected and transformed cells. Their ability to kill resides in a series of cytotoxic proteins stored in lysosome-related secretory granules in their cytoplasm. Those granules contain mainly the pore-forming protein perforin and a family of serine proteases termed granzymes which, upon target recognition and conjugate formation, are released in the target cells in order to induce apoptosis. Among the granzymes, granzyme A (GA) and granzyme B (GB) are the most abundant and the best characterized both in humans and mice. GA is a 50 kDa difulfide-bound homodimer and triggers cell death in a caspase-independent manner by inducing rapidly DNA fragmentation, nuclear envelope breakdown and reactive oxygen species (ROS) production. GA cleaves the endoplasmic-reticulum-associated complex inhibitor, termed SET-inhibitor, and therefore leads to DNA fragmentation mediated by exo- and endonucleases of the same complex. GA is also able to translocate into the nucleus, where it cleaves and inactivates DNA and nuclear integrity committed proteins. Driven by the mitochondrial membrane potential, GA enters mitochondria and cleaves the NDUFS3 subunits of the ETC complex I, leading to ROS production and cell death. GB, a 32 kDA protein, induces target cell apoptosis by both caspase- dependent and caspase-independent pathways. First, GB is able to cleave directly caspase-3, - 7, -8 and -10 and to initiate the caspase cascade. GB shares with the caspases some target proteins such as PARP-1, ICAD, lamin B cleaving them following aspartic acid residues similarly to caspase-3. A mitochondrial death pathway is also activated by GB in a caspase-independent fashion through cleavage of Bid, subsequent Bax-Bak oligomerization and mitochondrial outer membrane permeabilization (MOMP). The release of apoptogenic factors, like cytochrome c, AIF, Smac/DIABLO upon MOMP leads to a further activation of caspases and to an enhancement of the death insult. Similarly to GA, GB is able to translocate into the mitochondrial matrix in a Δψ-sensitive manner where it cleaves some subunits of the ETC complex I. NDUFV1, NDUFS1 and NDUFS2 cleavage leads to the impairment of the mitochondrial respiration and therefore to ROS production. The direct action of GA and GB in the mitochondria shows that both granzymes actively cross the outer and the inner membrane to reach their substrates by a mechanism that is most likely shared by all the other granzymes.

Neither GA nor GB possess any known mitochondrial targeting signal. Therefore we asked how those proteins could reach the matrix. My PhD work aimed to characterize the molecular mechanism by which GB (but also GA and caspase 3) enters the mitochondria to cleave ETC

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complex I subunits and produce ROS-dependent cell death. We examined granzymes’ primary sequences and 3D-structures and we found that the GB C-terminal α-helix alone was able to drive the import of the protein in the mitochondria. This α-helical C-terminal domain of GB is structurally conserved in all the granzymes and accross species having a series of basic residues clustering at the solvent-facing interface. The positive charge on GB was already demonstrated to be important for GB uptake in the cytosol of the target cells upon immunological synapse formation and degranulation. We showed that basic residues were also critical for GB mitochondrial import and that their modification resulted in a less effective GB-mediated cell death in vitro as well as in vivo. The lack of a canonical mitochondrial targeting signal suggested that GB import did not followed the classical way. Indeed, our results unambiguously demonstrated that GB mitochondrial entry is independent of the canonical TOM and TIM complex. As a matter of fact, neither the Tom40 channel nor the Tim23 channel, which mediate the import of the vast majority of the mitochondrial proteins, were necessary to import GB. Instead, we found that Sam50 channel of the sorting and assembly machinery (SAM) promoted GB, GA and caspase-3 import across the mitochondrial outer membrane.

Similarly, Tim22 channel of the translocase of the inner membrane complex mediated GB and GA import up to the matrix. Furthermore, we showed that when GB was prevented to reach the mitochondrial matrix, ROS production and cell death was also impaired, supporting the unanticipated concept that GB mitochondrial entry is crucial in the context of CTL/NK cell- mediated killing. The uncanonical entry of the granzymes showed that another mitochondrial gate pathway exists which could be used by many other proteins to access the mitochondria in physiologic as well as in pathologic conditions.

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Chapter II

INTRODUCTION

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2.1. The immune system

The immune system is a complex machinery of cells, tissues and organs that protect the body from exogenous invaders (such as bacteria, virus, fungi) and from endogenous factors produced by infected or altered cells (such as damaged tissues and cancer cells). The principal aim of the immune system is then to defend the organism and maintain the physiological homeostasis within it by two main mechanisms: the innate and adaptive response. The cellular players of the immune system originate all from hematopoietic stem cells through a developmental process called hematopoiesis (Golub et al. 2013). Hematopoiesis starts very early in embryo in the yolk sac and fetal liver to shift then to the spleen. Only with bone development (between 4^th and 5^th month of the fetal life), the hematopoiesis takes definitively place in the bone marrow. The hematopoietic multipotent stem cells, upon few early steps of differentiation, originate erythroid, myeloid or lymphoid progenitors.

Figure 1: Hematopoietic stem cells (HSCs) are multipotent stem cells and give rise to all blood cells, including immune cells.

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The erythroid lineage, derived from a common myeloid progenitor, gives rise to erythrocytes and megakaryocytes, the latter being the cells producing platelets. From the myeloid precursor originate granulocytes (neutrophils, eosinophils and basophils), macrophages, dendritic cells and mast cells. Among these, macrophages and neutrophils give rise to the first action against pathogens (especially bacteria) in the innate immune response. Natural killer (NK) cells are also playing a role in the innate immune response, but they originate together with lymphocytes (B and T lymphocytes) from a lymphoid progenitor. NK cells are classified as large granular lymphocytes because of their large granular cytoplasm, while B and T lymphocytes are defined as small lymphocytes since they have almost no cytoplasm in their immature and inactive form. T and B lymphocytes are protagonists of the adaptive immune system, supported by dendritic cells and macrophages which internalize, process and present the antigens to trigger lymphocytes activation.

2.1.1. Innate immune system

The first line of defense, considered part of the innate immune system, is represented by the skin and mucous membranes that physically and chemically prevent on pathogens entry into the body. Parts of this first barrier are also ciliated cells filtering pathogens and particles, enzymes secreted by mucous membranes and the body flora that blocks some harmful pathogens. When pathogens find a way to enter the body by overpassing the first barriers, the immune system launch a response by activating specific cells that are first players of the innate response. This response needs a period of few hours to several days to become completely effective. The main players of the innate response are phagocytic cells, such as macrophages and neutrophils that literally eat and digest microorganisms, especially bacteria. Those cells are known to recognize pathogens through pattern-recognition receptors (PRRs) that recognize and bind specific pathogens factors, called PAMPs (pathogen-associated molecular patterns) (Kumar et al. 2011). The best characterized PRRs are the Toll-like receptors (TLRs), first discovered in Drosophila having a role in embryonic development (Anderson et al. 1985) and only after as an indispensable sensor of pathogen molecules and initiator of the innate immune response in many organisms, including humans. Natural killer (NK) cells are also of innate immune cells able to kill infected or malignant cells.

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- 11 - a) Macrophages

Macrophages are cells of the innate immune system specialized in phagocytosis. They participate in the clearing of pathogens and dead cells and in the inflammatory response but they are also important for morphogenesis during embryonic development. Beside that, macrophages participate in antigen presentation to T-cells in the secondary lymphoid organs, bridging innate and adaptive immune response. Macrophages are present all over the body and originate from monocytes, large white blood cells derived from precursor cells in the bone marrow. Monocytes circulate in the bloodstream and in response to infection or tissue injury can migrate into the tissues where they differentiate in macrophages thanks to differentiation factors. The entry of monocytes (and generally of all the leukocytes) into the tissue is a process known as chemotaxis (Jones, 2000). This process is mediated by microbial particles, complement fragments and pro-inflammatory chemokines that functions as chemoattractants for monocytes. Upon monocyte differentiation into macrophage, the gene expression profile is also changing. Unlike monocytes, MHC-II expression considerably increases and macrophages that have phagocytosed the pathogen or the apoptotic cells and processed the antigen can activate CD8⁺ T-cells. Activated T-cells produce IFNγ and TNFα that further activate macrophages, enhancing their phagocytic activity. Macrophages possess a great variety of receptors by which they can recognize pathogens or their particles. Opsonic receptors, such as complement receptor or Fc receptors together with scavenger receptors can mediate macrophage adhesion and chemotaxis but also pathogen recognition and phagocytosis. The primary macrophage immune recognition process is mediated by PRRs (Mogensen, 2009) which recognized pathogen-associated molecular pattern (PAMPs). PAMPs include microbial components such as lipopolysaccharide (LPS), bacterial flagellin, mannose residues, unmethylated CpG and nucleotide variants typical of virus and bacteria. In addition to pathogen specific patterns, PRRs can also recognize host signals deriving from stressed or dead cells, termed damage-associated molecular patterns (DAMPs). In all the cases, PRRs are activated and trigger an early and massive response through many intracellular pathways.

PRRs are classified based on their cell localization in membrane-associated PRR and cytoplasmic PRR, but also depending on the microbial ligands they can bind. Toll-like receptors (TLRs) are the most estensively studied, but we have also retinoic acid-inducible gene (RIG)-I- like receptors (RIGs), NOD-like receptors (NLR) and C-type lectin receptors (CLR). All of them

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are not exclusive of macrophages but they are also present on densdritic cells (DCs) and other cells.

Table 1 (Takeuchi et al. 2010)

Ten different TLR have been identified in human sensing a great variety of pathogens because of their varied ligand specificity and their different localization. TLR1, TLR2, TLR4, TLR5 and TLR6 are located at the plasma membrane, whereas TLR3, TLR7, TLR8 and TLR9 are in the endosomal membranes where they will most likely meet they respective ligand. They are all type I transmembrane proteins with a leucine-rich N-terminal domain which binds PAMPs, a transmembrane domain and an IL1R homologous domain called Toll/IL1R or TIR domain. TLRs located at the plasma membrane bind extracellular pathogen molecules, mainly LPS and lipoproteins. Whereas, the endosomal located TLRs recognize mainly nucleic acids from bacteria and virus but also from the cells themselves in the context of stress.

TLR activation initiates a series of signaling pathways depending on their localization and on the adaptor molecules which are recruited to the TIR domain. The main activated pathways are NF-κB and MAPK pathways which induce a proinflammatory response and IFN regulatory factors (IRFs) expression. The proinflammatory response triggers cytokines and chemokines expression, such as IL-1, IL-6, TNFα, IL-8 that activate and recruit other immune cells at the site of infection. As result, they provoke an enhanced innate response increasing phagocytosis and

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clearing of the insult as well as the upregulation of MHC receptors and co-receptors (CD80, CD86, CD40) for the activation of the adaptive immune response. In particular, DCs and macrophages at the site of infection can then migrate to the lymph node and activate pathogen-specific T lymphocytes (Pozzi et al. 2005). This support the idea that the innate and the adaptive response are closely related, orchestrating together an effective reaction to the threats.

b) Neutrophils

Neutrophils are the more abundant cells among the white blood cells and are also known as polymorphonucleated leukocytes due to the presence of several irregularly shaped nuclei.

They are short-lived killers of the innate immunity that maturate in the bone marrow before being released to reach the infected tissue through the blood flow through a process called extravasation. This process is composed of four steps, rolling adhesion, tight adhesion, diapesis and transmigration, and is determined by interactions among adhesion molecules on neutrophils and vascular endothelial surface. In particular, selectins (E- and P-selectin) (Larsen et al. 1992) on the interior wall of the vessels bind carbohydrates on neutrophil surface to slow them down (rolling adhesion) and integrins on neutrophils like CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) (Ding et al. 1999) bind their complementary endothelial receptors to stop neutrophils (tight adhesion). At this stage neutrophils can cross the endothelial surfaces of blood vessels thanks to immunoglobulin superfamily adhesion molecules (CAMs) (Abelda et al. 1994) expressed by both neutrophils and endothelial cells and digest the basement membrane thanks to proteases. As final step, neutrophils move toward the infected tissue following a chemotactic gradient of CXCL8. Upon activation of phagocytic and complement receptors, neutrophils phagocyte pathogens similarly to the macrophages (Segal et al. 2005).

Pathogens degradation takes place in the phagosomes that fuse with two types of granules:

azurophilic/primary granules and specific/secondary granules both containing peptides and proteins able to digest the engulfed microorganism. Particularly, the specific granules contain component of the NADPH oxidase complex that is assembled after fusion wih the phagosomes.

NADPH oxidase leads to a massive respiratory burst with the release of reactive oxygen species (ROS) immediately converted into hydrogen peroxide. The respiratory burst leads also to an increase of the pH up to 8.0 and the consequent activation of peptides and proteins contained in the phagosomes which digest and destroy the pathogens. After killing, neutrophils are not

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capable to reassemble their granules. Therefore they undergo apoptosis and are phagocytosed by the macrophages. Recently, neutrophils have been associated with tumors showing a controversial role in the context of cancer (Uride at al. 2015). Tumor-produced cytokines induce an increased number of neutrophils in the blood that are then recruited to the tumor microenvironment. Two types of tumor-associated neutrophils (TANs) have been described so far: the anti and the pro-tumor neutrophils, termed N1 and N2 respectively. N1 have proinflammatory and anti-tumor functions that reside in their ability to secrete cytokines and chemokines and therefore recruit other cells of the immune system. On the other hand, N2 have been associated with a poor diagnosis due to their role in angiogenesis, immune soppression, metastasis and therefore cancer progression (Swierczak et al. 2015). However, what make neutrophils become pro- or anti-tumor cells remains to be further elucidated.

c) NK cells

Natural killer cells are the lymphocytes of the innate immune system. They are large cells with cytotoxic granules in the cytoplasm and circulate in a partially activated state. For that reason, they become rapidly effective against infections. NK cells move into the infected tissues in response to inflammatory cytokines binding their specific receptors on the NK cell surface.

Macrophages and dendritic cells participate to the NK cell activation through IL-12 and TNFα production triggering the production of type II interferon (mainly IFNγ) by NK cells. Thus, NK cells signal back in a positive activating loop toward macrophages, dendritic cells and T-cells, triggering the amplification of both the innate and adaptive immune response (Moretta et al.

2005; Walzer et al. 2005). On the contrary, type I interferons favor the amplification of the killer functions in NK cells. In particular, IFNα and IFNβ interfere with viral replication in all the cells and increase the expression of activating receptors on NK cells promoting their cytotoxic action against virus-infected cells. Unlike B- and T-cells, NK cells are lymphocytes of the innate immune system which do not have any antigen-specific receptor (Herberman et al. 1985;

Trinchieri 1995) but they express a repertoire of inhibitory and activating receptors. NK receptors can be classified in: a) lectin-like receptors (CD94 and NKG2), b) immunoglobulin-like transcripts (ILTs also called LILRs), and c) killer immunoglobulin-like receptors (KIRs). CD94 molecules and NKG2 form heterodimeric complexes that can be activators or inhibitors depending on the NKG2 type. Instead, in KIRs, the size of the intracellular domain can discriminate between activator (short cytoplasmic domain) and inhibitor (large cytoplasmic

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domain). The balance between inhibitory and activating signals dictates NK behavior. The best characterized mechanism by which NK could be activated or inhibited is the binding to MHC-I.

MHC-I, normally expressed on normal nucleated cells, is recognize and boud by the inhibitory NK receptors impeding their activation. Altered expression of MHC-I can induce NK to recognize those cells as infected and thereby lead to NK–mediated killing (Moretta et al. 1996).

d) Complement system

The complement system is made of more than 30 plasma proteins circulating in the body or located in specific tissues. Those proteins, coating bacteria and virus particles, facilitate the phagocytosis of such pathogens. Many of the proteins of the complement system are proteases that circulate as immature pro-enzymes known as zymogens and are activated by proteolytic cleavage. There are three different pathways by which complement proteins are activated triggering an amplification of the inflammation and a better phagocytic response: the alternative pathway and the lectin pathway that are part of the innate immune system, and the classical pathway that can also be activated in an adaptive immune response (Dunkelberger et al. 2010). Whatever the pathway, the activation of C3 fragment of complement represents a key step. Indeed, all the pathways lead to C3 activation and massive C3 cleavage into a small C3a fragment and a large C3b fragment. C3a is a soluble fragment that amplifies the inflammatory response by functioning as chemoattractant for effector cells.

Instead, C3b is covalently bound to the pathogen’s surface (complement fixation), being a tag for phagocytes to be recruited and to kill the pathogen. C3 binding to the pathogen is due to the exposure of a thioester bond that is hydrolysed by nucleophilic attack by amino and hydroxyl groups from pathogen’s surface macromolecules. On the other hand, the release of C3 in aqueous solution causes a conformational change making the thioester bond available for hydrolysis without any need of C3 cleavage. The hydrolysis of C3 to iC3 is the first step of the alternative pathway. The alternative pathway is the first response of the innate immune system in the case of a bacterial infection and is considered to be independent from any pathogen-binding initiator protein. The auto-hydrolyzed C3 (iC3) binds factor B that is then cleaved by factor D producing a small and a large fragment, Ba and Bb, respectively. While Ba is released, Bb stays attached to iC3 forming iC3Bb, a soluble C3 convertase. Thanks to Bb protease activity, iC3 is cleaved in C3a and C3b fragment. C3b bound to the pathogen surface leads to factor B cleavage by factor D and therefore to the formation of C3bBb alternative C3

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convertase. Once the alternative pathway is activated, macrophages can recognize bacteria trough the so-called complement receptors such as CR1, CR3 and CR4 that can bind either C3b or iC3b fragments on microbial surface. On the other hand, the complement cascade can proceed following C3b bond to the alternative C3 convertase, called the alternative C5 convertase. C5 is then converted in C5a and C5b fragments, with C5b being the initiator of the membrane-attack complex (MAC) together with C6, C7, C8 and C9 (Cole at al. 2003). In detail, C5b binds C6 and C7 allowing C7 to be inserted into the membrane. C8 also binds to C5b and initiates C9 polymerization and the formation of transmembrane pores. As C3a, C5a is soluble fragment that increases the inflammation process at the site of infection. Both molecules induce vascular permeability and increase blood flow resulting in the migration and accumulation of complement and other plasma proteins, neutrophils and monocytes in the tissue where infection took place. C3a and C5a are also called anaphylatoxins for their capacity to induce sometimes anaphylactic shock, an acute inflammatory response. The second pathway of complement activation is known as lectin pathway and is initiated by mannose- binding lectin (MBL) proteins. Mannose-containing carbohydrates on bacteria and viruses are recognized by MBL proteins and two associated serine proteases (MASP-1 and MASP-2).

Subsequently, C4 and C2 complement proteins are cleaved. C4b and C2a large fragments bind to the pathogen surface forming a C4bC2a complex that is the C3 convertase of the lectin pathway. C4a, the smaller C4 fragment, works as anaphylatoxin while C2b small fragment is inactive. The classical pathway activation can take place during either innate or adaptive immune response when C1 binds the C-reactive protein or an antibody on the pathogen surface, respectively. C1 complement protein is made of two zymogens, C1r and C1s with a structure similar to MASP-1 and -2 serine proteases, and one lectin protein C1q. C1s, cleaved and activated by C1r, leads to the cleavage of C4 and C2 and to the formation of the C3 convertase C4bC2a as in the lectin pathway. All together, the complement pathways signal the presence of pathogens in a specific compartment of the body through C3b formation and pathogen opsonization. Some other plasma proteins such as properdin D (or factor P) amplify the complement activation by stabilizing C3bBb on pathogen surface. This amplification loop of C3 cleavage is of course equilibrated by complement control proteins such as factor H and factor I that reduce the number of C3 convertase on microbial surface. Another family of complement control proteins consists of human cell plasma proteins that interfere with

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complement activation, such as DAF (decay-accelerating factor) and MCP (membrane co-factor protein) that cause C3 convertase dissociation or inactivation by factor I (Kim et al. 2006).

Figure 2: Activation pathway of the complement system. The three pathways are initiated by different molecules, but they all converge on the C3 fragment activation. Proteolytic cleavage of other complement proteins downstream of C3 finally leads to inflammation (C2b, C4a, C3a and C5a), opsonization (C3b and iC3b) and cell lysis (MAC complex: C5b, C6-9).

2.1.2. Adaptive immune system

The adaptive immune system is so called because it is specifically adapted to the pathogen against which it is directed. Two types of adaptive immune responses are distinguished: the humoral immunity and the cellular immunity, mediated by antibody-producing B lymphocytes and T lymphocytes, respectively. Unlike the innate immune system, the adaptive response needs more time to be launched since it takes 4-5 days for the lymphocytes to be activated.

The innate immune system actively works to manage the infection but it also contributes to the adaptive response initiation to finally ward off the threat.

B- and T-lymphocytes both originate in the bone marrow where B-cells continue their maturation, while T-cells migrate to the thymus. Every organism has his own endowment of specific lymphocytes, each with a unique receptor that could be an immunoglobulin in the case

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of B lymphocytes or a TCR for the T lymphocytes. B- and T-cell receptors are structurally related with some main differences. The B-cell receptors consist in a membrane bound immunoglobulins expressed on the surface of B lymphocytes whose secreted form gives the soluble form (antibodies). T-cell receptors are uniquely surface proteins. Immunoglobulins are formed by two large (heavy chains) and two small (light chains) polypeptides, all containing both constant and variable regions. TCRs consist of α and β chains (TCRα and TCRβ) having a constant and a variable region, too. The high variability in the antigen binding sites of B- and T- cell receptors is due to differences in the amino acid sequence. Indeed, genes encoding for the receptors are made of several genetic segments. The enormous diversity in the variable parts is given by the gene rearrangement mechanism, better known as somatic recombination. This mechanism was first elucidated in 1976 by Tonegawa (Hozumiet al. 1976) during B lymphocytes development in the bone marrow, for which he was awarded the Nobel Prize in Physiology or Medicine in 1978. The same was later showed for T lymphocyte receptors.

During B- and T-cell development, self-reactive lymphocytes, responsive to healthy tissues of the body, can arise. To avoid this, lymphocytes are selected through a mechanism known as immunological (or central) tolerance which takes place in the primary lymphoid organs. In particular, T lymphocytes encounter a positive selection in order to maintain only T-cells which are able to recognize self MHC molecules in the body. At this point, it is also determined the fate of a double positive T-cells to become CD4⁺ or CD8⁺ T-cell. On the surviving T-cells, a negative selection is then necessary to eliminate those which have a strong affinity with MHC- self peptide. The negative selection on T-cells has also a selective effect on B cells since their activation depends on CD4⁺ helper T–cells carrying the same antigen. Another process of the central tolerance, better characterized in B cell development, is the receptor editing which re- engages gene rearrangement of the autoreactive-antigen receptor without killing the cells.

Some self-reactive lymphocytes can still escape the immunological tolerance and leave the primary lymphoid organs. In the circulation and in the rest of the body other mechanisms of tolerance take place, called peripheral tolerance. The main players against autoreactive cells in the periphery are effector CD4⁺ T-cells called regulatory T-cells (T_reg). This subset of T-cells is distinguished by the expression of CD25 on their cell surface and the transcription factor FoxP3. Once activated by binding the specific antigen-MHC class II complex, Treg cells contact naïve self-reactive T lymphocytes and suppress their proliferation with inhibitory cytokines.

Auto-reactive B- and T-cells that arrive in the periphery enter into an unresponsive state called

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anergy caused by the exposure to circulating antigen in the absence of co-stimulation. Unlike the innate immunity, an adaptive immune response is not initiated in the site of infection but in dedicated tissues called secondary lymphoid organs, such as lymph nodes, spleen and gut.

Pathogens or their antigenic debris are carried by macrophages and dendritic cells through the draining lymph and meet circulating naïve B- and T-lymphocytes in the lymph nodes. Those lymphocytes which recognize their specific antigen will be selectively activated and will proliferate. Antigen recognition by the T-cell receptor is mediated by some glycoproteins on the antigen-presenting cells (APC) called major histocompatibility complex (MHC) molecules.

MHC molecules are not specific for a single antigen, but they are still selective for some peptide types. For this reason, several MHC molecules are expressed in the same antigen- presenting cell. Each MHC form is encoded by a single gene that is usually highly polymorphic among the population. Despite their large polymorphism, MHC molecules are commonly divided in two classes: MHC class I for antigens derived from intracellular pathogens and MHC class II for extracellular agents. The two classes of MHC molecules define also the type of T lymphocytes that can be activated by dendritic cells. For example, cytotoxic CD8⁺ T-cells, which are effective against intracellular infection, recognize peptides presented at the cell surface by MHC class I molecules. Whereas CD4⁺T lymphocytes function in the response to extracellular pathogens and they are activated by dendritic cells presenting antigen-MHC class II complex on their cell surface. MHC class II molecules are synthetized in the endoplasmic reticulum as MHC class I molecules, but they bind peptides in the endocytic vesicles where the extracellular pathogens are degraded upon endocytosis. The interaction with the antigen receptor of CD4⁺ T-cells takes place on the cell surface and stimulates T-cells to proliferate and differentiate in several types of helper T-cells (or effector CD4⁺ T-cells). Helper T-cells can migrate to the infected tissue and specifically target macrophages which have ingested pathogens. Moreover, they are able to secrete cytokines and attract other neutrophils and monocytes to the site of infection. Instead, another population of helper T-cells remains in the secondary lymphoid organs where they have been activated by dendritic cells. There, they specifically interact with B cells presenting the same antigen-MHC class II complex and drive them to proliferate and become antibody-producing plasma cells. Antibodies from B lymphocytes can work by many mechanisms, such as neutralizing pathogens by binding at them and impeding them to enter the cells and replicate, opsonizing the pathogens to make them engulfed by phagocytes, and activating the complement system. As stated earlier, lymphocyte activation and proliferation

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take several days to be completed. Once infection has been removed and no other antigens are circulating in the body, effector cells are destroyed except for some of them that become memory cells, able to recognize the same pathogen in case of re-infections. On memory cells resides the ability of the adaptive immune system to respond faster and in a more potent way to the same pathogens. Those long-lived memory cells guarantee the so-called immunological memory that represents also the basis for vaccination.

2.2. Cell-mediated cytotoxicity: CTL and NK cells

Cytotoxic T lymphocytes (CTL) and Natural Killer cells (NK) are the main players of the immune system against transformed and virus-infected cells. Both cell types are originated from the same lymphoid progenitors in the bone marrow and share the same mechanism of killing.

However, CTL and NK are part of the adaptive and innate immune system, respectively. As described before, T lymphocytes differentiate in the thymus and then mature in two subsets of T-cells depending on the expression on their surface of either CD4 or CD8 receptor. The T-cell commitment is tightly regulated by several transcription factors. Among them, Th cell transcription factor (ThPOK) leads to CD4 expression and cooperates with additional transcription factors to drive CD4⁺ T-cells differentiation into Th subset or regulatory T-cells (He et al. 2005). On the other hand, RUNX3 transcription factor downregulates CD4 expression and promotes differentiation into CTL (Taniuchi et al. 2002). In particular, cytotoxic T lymphocytes mainly express CD8 (CD8⁺ CTL) that works as co-receptor for the binding of the major histocompatibility complex class I (MHC-I) on the antigen-presenting cells with the T cell receptor (Engleman et al. 1981; Meuer et al. 1982). Another small subset of T lymphocytes that express CD4 (CD4⁺ CTL) has been found (Fleischer 1984) to express CTL-related genes, such as granzyme B, IFN-γ, and to possess cytotoxic properties (Takeuchi et al. 2015). Effector T-cells show higher expression of surface CD4 or CD8 and also an increased number of adhesion molecules such as CD2 and LFA-1 in order to interact with ICAM-1 and LFA-3 on target cells. CTLs and NK cells can kill target cells in two different ways: by activating the death receptor pathways or by granule exocytosis. While CTL can activate both pathways, NK cells seem to preferentially kill through the granule-dependent one. Both cell death pathways require a physical interaction between effectors and target cells.

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2.2.1. Lymphocyte receptor-mediated cell death pathways

Receptor-mediated cytotoxicity was first described in 1993 by Rouvier et al. (Rouvier et al., 1993) as a CTL alternative pathway to lysate target cells cultured in Ca²⁺-free condition where Fas was showed to be involved in the initiation of the death process. Similarly, few years after, the existence of the death-receptor pathway was also demonstrated in NK cell and again a Fas- FasL (as well as TRAIL/TRAIL-receptor or TNF/TNFR) interaction was confirmed to initiate target cell death (Montel et al., 1995; Zamai et al. 1998). Fas receptor (also called CD95 or APO-1) belongs to the Tumor Necrosis Factor Receptor (TNFR) gene superfamily, as well as the receptors for TNFα, TRAIL, and CD40. Fas receptor is expressed in many tissues and cell lines as a 45 kDa type I membrane protein characterized by a cysteine-rich extracellular domains. In particular, Fas is strongly expressed in double positive CD4⁺ CD8⁺ thymocytes suggesting a role in clonal deletion in the thymus (Yonehara et al. 1994). Fas expression can be increased upon lymphocyte activation but also by cytokines. Fas-FasL (or TNF/TNFreceptor) system is capable to trigger the activation-induced cell death mechanism (AICD) in activated and auto-reactive T- cells in the periphery of the body. It has been shown that in mature T-cells the expression of FasL is high in order to induce apoptosis in Fas-expressing lymphocytes. For those cells, AICD results from repeated self-antigen stimulation as a form of peripheral tolerance of T lymphocytes (Alderson et al. 1995). As for T lymphocytes, anergic autoreactive B cells undergo CD4⁺ T cell-mediated apoptosis in a Fas-dependent manner (Rathmell et al. 1995). The latter was shown by several studies in which mutations of Fas receptor (lpr) or Fas ligand (gld) result

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in lymphoproliferation and autoimmune disorders in mice and humans (Roths et al. 1984;

Watanabe-Fukunaga et al. 1992; Drappa et al. 1996).

FasL (CD95L) is a 40 kDa molecule, member of the TNF superfamily and is expressed by the effector CTLs and NK cells. FasL (as for TNF-α and TRAIL) is present both as a membrane-bound (mFas) and as soluble protein (sFas) upon metalloprotease-mediated cleavage, but only the membrane-bound form is able to induce apoptosis in mouse (O' Reilly et al. 2009). On the contrary, it has been shown that sFasL participates to other cell death-unrelated pathways, such as inflammatory response (though activation of NF-κB pathway) (Ponton et al. 1996), cell differentiation and proliferation. In the contest of CTL and NK cytotoxicity, the death receptor on target cells is bound by effector cells expressing ligands of the tumor necrosis factor family on their surface. In particular, Fas/FasL binding (as well as TRAIL/TRAIL-receptor) leads to receptor trimerization on the target cell and recruitment of the Fas-associated death domain (FADD or Mort-1). Fas-FADD binding is due to death effector domain (DED) interactions between the Fas intracellular portion and at the C-termini of FADD. The N-termini death domain of FADD recruits initiator procaspase-8 (and 10 in humans), forming all together the death-inducing signaling complex (DISC). Caspase-8 and caspase-10 inhibitory proteins (FLICE1 and FLICE2) have been identified in virus and human as vFLIP and cFLIP, respectively. They contain DED and a caspase-like domain and could compete for the binding to FADD. The initiator procaspases-8 homo-oligomerizes and becomes fully activated by autoproteolytic processing. Fully active initiator caspases are then released in the cytosol as heterotetramer (two small p10 subunits and two large p20 subunits) in order to target effector caspases (caspase-3 and caspase-7) and the pro-apoptotic BH3-only protein Bid, member of the Bcl-2 protein family. The truncated and active form of Bid (tBid) activates Bax/Bak which mediate mitochondrial outer membrane permeabilization (MOMP) and release of apoptogenic factors such as cytochrome c, Smac/DIABLO, Omi/Htra2. Cytochrome c enhances Fas-induced cell death triggering the formation of the apoptosome and Apaf-1-mediated activation of caspase- 9. Moreover, Smac/DIABLO removes XIAP inhibitory effect on effector caspases. The mitochondrial intrinsic pathway is taking place in the so-called type II cells through tBid translocation. In those cells, the activation of procaspase-8/10 is not robust enough to provide an effective cell death as for type I cells. Indeed, mitochondrial apoptogenic factor release provides a positive feedback loop to the effector caspase-3/7 through caspase-9 activation.

Lately, it has been strongly disputed the role of caspase-8 in typeI/II cell classification and XIAP

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have been pointed out as critical factors, since XIAP inactivation is able to shift type II into type I cells (Hao et al. 2010).

Figure 4: Receptor-mediated cell death pathways. The activation of the death receptors on the plasma membrane leads to the formation of the DISC complex. Caspase-8 in the DISC complex is activated by auto-proteolytic processing and cleaves effector caspases (Caspase-3 and -7). In type II cells, the intrinsic pathway is also activated through cleavage of Bid with the release of mitochondrial apoptogenic proteins (Rodriguez et al. 2015).

2.2.2. Granule-exocytosis pathway

The granule-dependent pathway is the major mechanism of CTL/NK cell-mediated killing of virus infected and transformed cells. The cytotoxic granules in NK cells are formed during development, whereas CTLs need to be activated in order to assemble these secretory organelles. Granules contain mainly perforin and a family of serine proteases known as granzymes which are delivered into the target cells in order to mediate cell death. Perforin is a 60-70 kDa glycoprotein with the ability to polymerize and form pores in the lipid membrane of the target cells. Similar to perforin, granulysin (GNLY) is another pore-forming protein with a high cytolytic activity against bacteria and tumor cells (Peña et al. 1997; Stenger et al. 1998). It is a member of the saposin-like protein (SAPLIP) family and it has been found exclusively in the cytotoxic granules of human, but not rodent cells. It has been demonstrated that granulysin

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could deliver granzymes in the bacteria infecting the target cells such as M. tuberculosum.

Once in the bacteria, granzymes cleave superoxide dismutases (SODs) and catalases making the parasites more prone to die for oxidative stress (Walch et al. 2014). Granzymes are proteases with different substrate specificity that trigger target cell apoptosis by activating several pathways. The most abundant granzymes in mouse and human are GA and GB which have also been better characterized. In addition to these proteins, granules contain also some lysosome-associated membrane proteins, such as Lamp-1, Lamp-2 and CD63, and lysosomal enzymes (hydrolases), including cathepsin-B, -C and –D, acid phosphatase, etc. For all these reasons, the cytotoxic granules are considered as lysosome-related organelles (LRO_s) having features of secretory organelles (Peters et al. 1991). The acidic pH in the granule environment prevents perforin and granzymes to be activated and favors their tight binding and packaging with serglycin, a negatively charged proteoglycan matrix protein. Similarly, calreticulin binds and inhibits perforin in a Ca²⁺-dependent manner by preventing perforin polymerization into the granules and killer cell autolysis. Granzymes are processed to their final active form by cathepsin C, while perforin is activated in the granules by a cysteine protease. CTLs and NK cells are also protected by cathepsin-B contained in the cytotoxic granules, which covers and protect the plasma membrane of killer cells from perforin action and granzyme B entry upon granule exocytosis. Upon recognition and conjugate formation with a target cell, CTL/NK trigger granule migration toward the immunological synapse (IS) where the granule content is released to initiate a death response in the target cells. Synapse formation is accompanied by MTOC (microtubule organizing center) polarization and granule movement along microtubules to the presynaptic membrane in the effector cells. In details, MTOC translocates and docks the plasma membrane at the central supramolecular activation complex (cSMAC), a typical circular structure due to the accumulation of the adaptor protein talin (cytoskeleton linker) and adhesion molecules at the killer/target-cell contact site. Actin and actin-motor protein, myosin II A, are then required for cytotoxic granule delivery to the synaptic cleft (Sancho et al. 2002).

Granule content is then released in the immunological synapse by exocytosis (or degranulation). Here, perforin and granzymes interact with the target cell to induce apoptosis.

Degranulation leads to the exposure of lysosome-related glycoproteins such as CD107a/b and CD63 on the cell surface. Recently, it has been demonstrated that mechanical force at the immunological synapse enhances perforin pore formation in a space- and time-dependent manner. The non muscle myosin II (NMII) is crucial for force exertion at the IS and its inhibition

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in CTL reduces cell cytotoxicity (Basu et al. 2016). Granzyme-induced cell death in the target cells is dependent on perforin. Indeed, mice deficient in perforin show immunodeficiency and impaired efficacy against viruses (Kägi et al. 1994; Lowin et al. 1994). The original model for perforin-mediated delivery of granzymes into the target cells suggests that perforin forms pores in the plasma membrane and let granzymes enter into the cytosol. However, perforin pores in the plasma membrane have a cut-off too small to let granzymes pass through when perforin is used at sublytic concentration. Whereas, cells treated with higher doses of perforin show pores large enough for granzymes to pass but the cells are preferentially killed by necrosis. Later, it has been demonstrated that the lack of perforin does not prevent granzyme B uptake, but impairs granzyme-mediated apoptosis (Shi et al. 1997). Based on these evidences, a new model was proposed in which perforin pores on the plasma membrane allows a Ca²⁺ influx and granzymes and perforin are co-endocyted by the target cells as result of membrane repair response mechanism (Reddy et al. 2001; Keefe et al. 2005). Granzymes and perforin endocytosis is a clathrin- and dynein-dependent mechanism and it leads to the formation of giant EEA-1⁺ early endosomes, named gigantosomes (Thiery et al. 2010).

Gigantosomes form in the target cells by fusion of Rab5-expressing early endosomes and are negative for the lysosomal marker LAMP-1 (CD107a) (Thiery et al. 2011). Granzymes are then released in the cytosol within few minutes (~15 minutes) thanks again to the action of perforin. By inhibiting clathrin- and dynein-dependent endocytosis, the target cell death shift from granzyme-mediated apoptosis to necrosis as for a perforin-damaged cell. As previously hypothesized by Froelich and his group, perforin can act as an endosomolysin forming pores in the gigantosome membrane and allowing granzyme release (Metkar et al. 2002). Consistently, perforin pore formation in the giant endosomes was assessed by confocal microscopy, confirming that granzyme release in the cytosol is perforin-mediated. Granzyme is slowly released already within 10 minutes, time for perforin to multimerize and form pores in the gigantosomes. After 15 minutes, perforin-mediated pores cause the collapse and the break of gigantosomes with the massive release of their cargo (Thiery et al. 2011).

2.2.3. Perforin structure and function

Perforin is one of the major components of the cytotoxic granules of CTLs and NK cells. It was first isolated in 1985 from murine CTLs and human NK cells and identified as a Ca²⁺-dependent pore forming protein thanks to its similarity to the C9 fragment of complement, another pore-

Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B

Thesis

Reference

Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B

UNIVERSITÉ DE GENÈVE

Département de biologie cellulaire FACULTÉ DE SCIENCES Professeur Jean-Claude Martinou

Département de physiologie et métabolisme

FACULTÉ DE MÉDECINE Professeur Denis Martinvalet

Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Valentina CHIUSOLO de Benevento (Italie)

Thèse n° - 4959 -

Genève

Atelier de reprographie à Uni Mail

2016

UNIVERSITÉ DE GENÈVE

Département de biologie cellulaire FACULTÉ DE SCIENCES Professeur Jean-Claude Martinou

Département de physiologie et métabolisme

FACULTÉ DE MÉDECINE Professeur Denis Martinvalet

Sam50 and Tim22: a novel mitochondrial gate for the import of Granzyme B

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Valentina CHIUSOLO de Benevento (Italie)

Thèse - 4959 -

Genève

Atelier de reprographie à Uni Mail

2016

Table of contents

List of figures and tables

List of Abbreviations

Chapter I

RÉSUMÉ AND SUMMARY

1.1. Résumé

1.2. Summary

Chapter II

INTRODUCTION

2.1. The immune system

2.2. Cell-mediated cytotoxicity: CTL and NK cells