TMX1 Preferentially Acts Upon Transmembrane Polypeptides

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Doctoral Thesis

In vivo characterization of TMX1: TMX1 preferentially acts upon transmembrane polypeptides

Author(s): Brambilla Pisoni, Giorgia

Publication Date: 2016

Permanent Link: https://doi.org/10.3929/ethz-a-010665921

Rights / License: In Copyright - Non-Commercial Use Permitted

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DISS. ETH NO. 23381

IN VIVO CHARACTERIZATION OF TMX1: TMX1 PREFERENTIALLY ACTS UPON TRANSMEMBRANE POLYPEPTIDES

A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich)

presented by GIORGIA BRAMBILLA PISONI

Laurea Magistrale in Biologia Molecolare della Cellula, Università degli Studi di Milano

born on 08.02.1986 citizen of Italy

accepted on the recommendation of Markus Aebi Maurizio Molinari Paola Picotti Roberto Sitia

2016

Parts of this thesis have been published in the following articles:

Noack, J., Brambilla Pisoni, G. & Molinari, M. Proteostasis: bad news and good news from the endoplasmic reticulum. Swiss medical weekly 144, w14001, doi:10.4414/smw.2014.14001 (2014).

Tannous, A., Brambilla Pisoni, G., Hebert, D. N. & Molinari, M. N-linked sugar-regulated protein folding and quality control in the ER. Seminars in Cell & Developmental Biology 41, 79-89, doi:10.1016/j.semcdb.2014.12.001 (2015).

Brambilla Pisoni, G., Ruddock, L. W., Bulleid, N. J. & Molinari, M. Division of labor among oxidoreductases: TMX1 preferentially acts on transmembrane polypeptides. Mol Biol Cell 26(19):3390-400. doi: 10.1091/mbc.E15-05-0321 (2015).

Brambilla Pisoni, G. & Molinari, M. Five Questions (with their Answers) on ER-associated Degradation. Traffic (2016).

Abstract

The endoplasmic reticulum (ER) is a complex organelle divided into different sub- compartments that are required for the execution of diverse functions, such as protein and lipid synthesis, Ca++ storage and drug detoxification. The rough ER (rER) is the subdomain designated for the production of secretory and membrane proteins in eukaryotic cells, which constitute about one third of the total proteome of the cell.

Protein translocation into the ER represents the first stage of protein secretion. Upon ER import, the vast majority of nascent polypeptides are modified by the oligosaccharyltransferase complex (OST), which catalyzes the addition of a pre-formed 14- subunits oligosaccharide (3 glucoses, 9 mannoses and 2 N-acetylglucosamines) specifically on the -N-X-S/T/C- consensus sequences displayed by the polypeptide chains. This process is called N-glycosylation and is responsible for both the increase in solubility of the polypeptides and the creation of binding motifs for ER resident lectins that drive glycopolypeptide folding.

Once the nascent polypeptide has been N-glycosylated, glucoses and several mannose residues are sequentially removed by ER-resident glycosyl hydrolases in a finely-regulated process known as glycan trimming which dictates the glycopolypeptide fate until its complete maturation or selection for degradation.

Maturing mono-glucosylated polypeptides enter the folding program principally managed by the ER lectins calnexin (CNX) and calreticulin (CRT). CNX and CRT in turn recruit specific ER resident enzymes, which assist folding rate-limiting steps. These are enzymes that catalyze the formation of native inter- and intra-disulfide bonds, the protein disulfide isomerases (PDI), and the isomerization of peptidyl-prolyl bonds, the peptidyl-prolyl isomerases (PPI).

After the release from the CNX-CRT folding cage, a sophisticated quality control machinery guarantees that only correctly folded polypeptides are able to leave the ER via COPII-

i mediated vesicles destined to the Golgi compartment. Polypeptide folding is an error prone process. If uncontrolled, accumulation of misfolded polypeptides can become extremely dangerous for the cell.

Specific sensors within the ER lumen judge whether misfolded proteins can re-enter the folding program for new folding attempts or must be labeled as terminally misfolded and addressed for ER-associated degradation (ERAD), a complex series of events that comprehend polypeptide recognition, linearization for retro-translocation, poly-ubiquitylation and finally degradation by cytosolic proteasomes.

The lumen of the mammalian ER contains more than 20 members of the PDI superfamily that intervene both during folding of newly synthesized polypeptides or during preparation for disposal of terminally misfolded ones. The reasons for the high redundancy of PDI members and the substrate features required for preferential engagement of one, or the other, are poorly understood.

To shed light into the PDI cellular roles and specificities, we focused our attention on the PDI subgroup called thioredoxin-related transmembrane proteins (TMX). We investigated how TMX1, one of the five transmembrane members of the TMX subfamily, intervenes during the folding program of model folding-competent substrates.

We found that TMX1 forms functional complexes with CNX and preferentially intervenes during maturation of cysteine-containing membrane-associated proteins, while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane.

Likewise, analysis of a possible involvement of TMX1 in the clearance of misfolded polypeptides from the ER revealed a selective intervention of the enzyme on membrane- bound proteins.

As such, these data demonstrate that TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.

Riassunto

Il reticolo endoplasmatico (ER) è un organello complesso diviso in differenti sotto- compartimenti che sono necessari per l'esecuzione delle diverse funzioni, come sintesi di proteine e lipidi, riserva di Ca2+ e detossificazione da droghe. L’ER rugoso (RER) è il sottodominio designato per la produzione di proteine secretorie e di membrana in cellule eucariotiche, le quali costituiscono circa un terzo del proteoma totale della cellula.

La traslocazione nell’ER della proteina rappresenta la prima fase della via secretoria. A seguito dell’importazione nell’ER, la stragrande maggioranza dei polipeptidi nascenti viene modificata dalla oligosaccaride trasferasi (OST), che catalizza l'aggiunta di un oligosaccaride costituito da 14-subunità (3 glucosi, 9 mannosi e 2 N-acetilglucosammine) specificamente sulle sequenze consenso –N-X-S/T/C- delle catene polipeptidiche. Questo processo è chiamato N-glicosilazione ed è responsabile sia dell'aumento della solubilità dei polipeptidi, che della creazione di motivi che vengono specificamente riconosciuti da lectine che risiedono nell’ER e che guidano il ripiegamento delle glicoproteine.

Una volta che il polipeptide nascente è stato N-glicosilato, i residui di glucosio e diversi mannosi vengono sequenzialmente rimossi da idrolasi dell’ER in un processo finemente regolamentato noto come trimming dei glicani che detta il destino della glicoproteina fino alla sua completa maturazione o fino alla sua degradazione.

La forma mono-glucosilata della proteina entra nel programma di ripiegamento, principalmente gestito dalle lectine calnexina (CNX) e calreticulina (CRT). CNX e CRT, a loro volta reclutano enzimi specifici che assistono alcune fasi rate-limiting del processo di ripiegamento proteico. Questi comprendono enzimi che catalizzano la formazione di ponti disolfuro, chiamati disolfuro isomerasi (PDI), e l'isomerizzazione dei legami peptidil-prolil, chiamati peptidil-prolil isomerasi (PPI).

Al termine del programma di ripiegamento supervisionato da CNX e CRT, un sofisticato apparato di controllo garantisce che solo i peptidi correttamente ripiegati siano in grado di

iii lasciare il reticolo tramite vescicole COPII destinate al compartimento di Golgi. Il ripiegamento delle proteine è un processo incline all’errore. Se tale processo diviene incontrollato, l’accumulo di peptidi mal ripiegati può costituire un pericolo reale per la sopravvivenza della cellula.

Sensori specifici all'interno del lume dell’ER giudicano se le proteine mal ripiegate possono rientrare nel programma di ripiegamento per un nuovo ciclo di folding o se devono essere etichettate come prodotti aberranti e indirizzati alla degradazione associata all’ER (ERAD), la quale prevede una complessa serie di eventi che comprende il riconoscimento del polipeptide, la sua linearizzazione per abilitarlo alla retro-traslocazione, la sua poli- ubiquitinazione e, infine, la sua degradazione ad opera dei proteasomi citosolici.

Il lume dell’ER delle cellule di mammifero contiene più di 20 membri facenti parte della superfamiglia delle PDI che intervengono sia durante il processo di ripiegamento delle proteine di nuova sintesi, sia durante la preparazione per la degradazione di peptidi mal ripiegati. La ragione che si cela dietro all'alto grado di ridondanza tra i membri delle PDI e la preferenza di questi per taluni specifici substrati, sono aspetti poco conosciuti.

Per fare luce sul ruolo e sulle diverse specificità esibite dalle PDI, in questo studio abbiamo focalizzato la nostra attenzione sulle PDI del sottogruppo TMX, il quale comprende cinque proteine legate alla membrana che presentano un dominio thioredoxin-like. Abbiamo studiato come TMX1, il primo membro della sottofamiglia TMX, interviene durante il programma di ripiegamento di proteine modello folding-competent.

In questo lavoro mostriamo che TMX1 forma complessi funzionali con CNX e preferenzialmente interviene durante la maturazione di proteine associate alla membrana e che contengono residui di cisteina, ignorando le stesse, se non ancorate alla membrana.

Analogamente, in uno studio volto all’identificazione di un ruolo di TMX1 nel processo di preparazione per la degradazione di peptidi mal ripiegati, abbiamo mostrato che TMX1 interviene selettivamente su proteine legate alla membrana.

Questi dati dimostrano che TMX1 rappresenta il primo esempio di catalizzatore redox in cellule viventi specifico per substrati che presentano una determinata topologia.

Abbreviations

A1AT: α-1-antitrypsin

BACE501: beta-secretase 1

CNX: calnexin

CRT: calreticulin

ER: endoplasmic reticulum

ERAD: ER-Associated Degradation

ERAD-C: ERAD substrate with cytosolic defect

ERAD-L: ERAD substrate with luminal defect

ERAD-LM: membrane-bound ERAD-L substrate

ERAD-LS: soluble ERAD-L substrate

ERAD-M: ERAD substrate with membrane defect

ERManI: ER α 1,2-mannosidase I

ERQC: ER quality control

GSH: glutathione

MLEC: malectin

NHK: null Hong Kong

OST: oligosaccharyl transferase

PDI: protein disulphide isomerases

PPI: peptidyl prolyl isomerases

TMX: thioredoxin-related transmembrane proteins

UGGT1: UDP-glucose:glycoprotein glucosyltransferase

UPR: unfolded protein response

UPS: ubiquitin proteasome system

Table of contents

1. Introduction ...... 1

1.1 The Endoplasmic Reticulum (ER) ...... 1 1.1.2 Sub-compartments of the ER ...... 1 1.1.2 Polypeptide entry into the ER ...... 2 1.1.3 N-glycosylation of the nascent polypeptides ...... 3 1.1.4 Substrate binding to malectin (MLEC) ...... 5 1.1.5 Substrate binding to calnexin (CNX) and calreticulin (CRT) ...... 7 1.1.6 Protein release from CNX and CRT ...... 7 1.1.7 Quality control (QC) ...... 8 1.1.8 Export of native proteins from the ER ...... 9 1.2 ER-Associated Degradation (ERAD)...... 9 1.2.1 N-glycan processing: the signal for degradation...... 9 1.2.2 Recognition of ERAD substrates...... 10 1.2.3 Preparation for dislocation across the ER membrane ...... 11 1.2.4 ER-to-cytosol polypeptide’s translocation, poly-ubiquitination and proteasomal degradation ...... 12 1.2.5 ERAD machineries are hijacked by pathogens ...... 13 1.3 ER stress and the unfolded protein response (UPR) ...... 14 1.4. Rate-limiting reactions during protein folding and ERAD ...... 15 1.4.1 Peptidyl-prolyl bonds isomerization ...... 15 1.4.2 Disulfide bonds formation, isomerization and reduction ...... 16 1.5 The PDI family ...... 19 1.5.1 The structure of PDI ...... 20 1.5.2 Select examples of PDI proteins...... 21 1.6 Mechanisms of action of PDI proteins ...... 27 2. Results ...... 29

2.1 TMX1, an ER stress non-responsive ER-resident member of the PDI superfamily ...... 29

2.2 TMX1 in vivo study: TMX1C/A trapping mutant ...... 29

2.3 Ectopically expressed TMX1 and TMX1C/A are ER-resident proteins ...... 30

2.4 TMX1C/A discriminates between substrates’ topologies ...... 31

2.4.1 TMX1C/A preferentially associates with membrane-bound substrates: evidences from mass spectrometry analysis ...... 31

2.4.2 TMX1C/A preferentially associates with membrane-bound substrates: evidences from biochemical analysis ...... 32

2.5 Role of TMX1C/A in protein folding ...... 34

2.5.1 TMX1C/A establishes mixed disulfides with newly synthesized membrane-bound BACE501 ...... 34 2.5.2 Client-mediated association between TMX1 and CNX ...... 37

2.5.3 Analysis of steady-state TMX1C/A:BACE501 mixed disulfides by WB ...... 39

2.5.4 TMX1C/A delays BACE501 maturation ...... 40 2.6 Ectopic expression of trapping mutants of PDI family members and BACE501 maturation ..... 42

2.6.1 Selective action of TMX1C/A on BACE501 ...... 42

2.6.2 TMX4C/A does not affect BACE501 maturation ...... 44

2.6.3 Selective action of TMX1C/A vs. non-selectivity of ERdj5C/A ...... 45

2.7 Role of TMX1C/A in ERAD ...... 46

2.7.1 TMX1C/A preferentially associates with membrane-bound folding-defective polypeptides ...... 46

2.7.2 TMX1C/A intervenes during BACE457 disposal ...... 48 3. Discussion ...... 52

3.1 PDI proteins: an overview ...... 53 3.2 In vitro characterization of PDI proteins ...... 54 3.2.1 Assays to detect oxidoreductase’s activity ...... 54 3.2.2 In vitro characterization of TMXs ...... 55 3.3 In vivo studies: a question of redundancy and specificity ...... 56 3.4 Functional characterization of TMX1 ...... 58 3.5 Functional evidences about the other TMX proteins ...... 60 4. Materials and methods ...... 62

Antibodies, expression plasmids, and inhibitors ...... 62 Cell lines and transient transfection ...... 62 Cell lysis and western blots ...... 63 Metabolic labeling, immunoprecipitations, and EndoH treatment ...... 63

vii

Indirect immunofluorescence ...... 64 RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) ...... 64 Quantitative real-time PCR (qRT-PCR) ...... 64 Mass spectrometry ...... 65 5. References ...... 66

6. Appendix ......

Curriculum vitae ...... PERSONAL DATA ...... EDUCATION ...... List of publications ...... RESEARCH ARTICLES ...... REVIEWS ...... Acknowledgements ......

viii

1.Introduction

1.1 The Endoplasmic Reticulum (ER)

1.1.2 Sub-compartments of the ER

The ER is a complex cellular organelle with a peculiar reticular morphology characterized by the presence of different sub-compartments that carry out diverse functions. The ER structure appears as a continuous membrane system delimiting a common intraluminal space 1,2. The ER has been historically subdivided into the smooth (sER) and the rough ER (rER) that form a network of tubular and sheet-like structures 2-6. Generally, sheets correspond to rER while tubules correspond to sER 7. ER sheets are defined as flat surfaces where the membrane can expand up to many micrometers with tiny curvatures. Instead, ER tubules are highly curved and highly crossed cylindrical structures 7. The relative amount of sER and rER depends on the cell-type and parallels the cellular function 8. Beyond the sER/rER subdivision, the ER comprehends different specialized domains such as the nuclear envelope (NE), the peripheral ER, the contact sites with other organelles and the ER exit sites 9. The ER structure shows a high degree of plasticity and is continuously remodeled according to the diverse cellular needs 10.

Generally, the sER shows a sponge-like architecture and consists of tubules that are more convoluted then the ones of the rER 11. The sER fulfills different cellular functions, such as steroid production 12, lipid synthesis 13,14, protein export 15 and drug metabolism 16,17. The sER also establishes contact sites with other cellular organelles such as mitochondria, peroxisomes, lysosomes, late endosomes, Golgi and the plasma membrane 8. Moreover, it serves as the major Ca2+ store of the cell 18-20. Interestingly, the majority of the ER resident chaperones is Ca2+-binding proteins and is responsible for the Ca2+-buffering in the ER. The contact sites between the mitochondria and the ER are particularly important to regulate Ca2+ levels: this close apposition is established at sER regions called mitochondria-associated membrane (MAM) 21,22. Beyond its role in calcium regulation, the MAM is also involved in other functions, such as lipid synthesis and immune response 23.

The rER, whose name derives from the presence of ribosomes bound to the cytosolic side of the ER membrane conferring the so-called “rough” aspect, possesses a sheet-like structure. It is the ER subdomain specifically designated for the production of about 7500 secretory and membrane proteins in mammalian cells, which constitute about one third of the total proteome of the cell 24,25.

1.1.2 Polypeptide entry into the ER

The majority of polypeptides addressed to the secretory pathway and to the cellular membranes displays an N-terminal signal peptide that consists of a short hydrophobic amino acid stretch. The first model that has been proposed, called “signal hypothesis” 26, implied the presence of a receptor for the recognition of the signal sequence located at the ER membrane. What has been discovered later on, is that the signal peptide binds to free cytosolic ribosome and upon initial translation engages the signal recognition particle (SRP), which targets the mRNA-ribosome-SPR complex to the SPR receptor close to the translocon complex in the ER membrane 27-30.

Alternatively, SRP-independent mechanisms do also exist, by which mRNAs encoding ER- resident proteins are targeted to the ER 31,32.

The translation of the polypeptide starts in the cytosol but it is then blocked until the polypeptide reaches the ER membrane 33. Here the translation re-initiates at the level of the ER translocon. The translocation complex has been described as an aqueous pore made of different proteins that span the ER bilayer 34,35. The constituents of that complex are the multi-spanning Sec61α, the single-pass Sec61β and Sec61γ, the signal peptidase complex, the translocating chain-associated membrane protein (TRAM), the translocon associated protein complex (TRAP), the oligosaccharyltransferase complex (OST) and other accessory proteins 36. Sec61α together with Sec61β and Sec61γ form a heterotrimeric complex, with the Sec61α transmembrane domains formally defining the pore cavity 37.

Upon arrival at the ER translocon, the signal peptide inserts into the Sec61 channel, inducing the repositioning of a short helix (helix 2) that occludes the pore and the consequent opening

2 of the channel 38-40. It was thought that the Sec61 channel’s open conformation was stabilized by the transit of the polypeptide chain itself 41. Recent evidences obtained by purification of the ribosome-Sec61 complex in association with the first 86 amino acids of the substrate preprolactin demonstrated that the opening of the translocation channel is due to the recognition and insertion of the substrate’s signal sequence 40. This is because the hydrophobic signal peptide replaces the occluding helix 2, stabilizing the open conformation of the channel. Co-translational translocation is unidirectional and is driven by both the elongation of the polypeptide chain and the hydrolysis of GTP 42.

The signal peptide is removed by the signal peptidase complex 43. The cleavage rate differs from protein to protein and may influence the topology, the functionality and the folding efficiency of the protein. Moreover, the cleavage efficiency has been found to be linked with several diseases 44. The ER degradation-enhancing alpha-mannosidase-like protein 1 (EDEM1) is a well-known example of cleavage-regulated protein topology. In fact, it can give rise to a soluble protein when the signal sequence is cleaved or can originate a membrane bound protein when the signal sequence is retained 45. In other cases, as for the glycoprotein gp160 of the human immunodeficiency virus (HIV), the signal peptide cleavage takes longer in order to facilitate the glycoprotein’s folding 46.

1.1.3 N-glycosylation of the nascent polypeptides

Most proteins entering the ER lumen possess -Asn-Xaa≠Pro-Ser/Thr/Cys- sequons within the polypeptide chain 47,48. The side chain of asparagine residues in these consensus sequences is rapidly modified through the covalent attachment of a pre-formed oligosaccharide made of three glucoses, nine mannoses and two N-acetylglucosamines (Glc3Man9GlcNAc2-) (Fig. 1) 49,50. The addition of this 14-subunits oligosaccharide is catalyzed by the OST complex 51-54. Mammalian cells possess two different OST complex types, known as STT3A and STT3B 55. They catalyze the co-translational and the post-translational glycosylation of nascent polypeptides, respectively 53,56. Each OST complex consists of one catalytic subunit (STT3A or STT3B) and other six proteins common to both complexes that are ribophorin I, ribophorin II, OST48, DAD1, OST4 and MagT1 or Tusc3. There are evidences that every

3 subunit of the OST complex confers glycosylation efficiency towards specific polypeptide subsets. Ribophorin I, for example, has been shown to be crucial for the proper glycosylation of select single-spanning membrane polypeptides 57-59. Moreover, each subunit of the OST complex cooperates with the others to confer complex’ stability 60,61.

Figure 1. N-glycan composition and processing. The pre-formed oligosaccharide is composed of three glucoses (dark gray circles), nine mannoses (light gray circles) and two N-acetylglucosamines (black squares). The oligosaccharide branches are shown with A, B and C. The oligosaccharide processing enzymes are listed in the figure and their action is shown. The type of glycosidic bond is shown in color (from 62). Symbol and color code for glycan representation: Varki et al., 2009 Proteomics.

N-glycosylation increases the solubility of the newly synthesized polypeptides 49. Moreover, the processing of the protein-bound oligosaccharide generates oligosaccharide-trimming intermediates that recruit ER-resident lectins thereby dictating glycopolypeptide’s folding, quality control, export or degradation 62-64. Outside the ER, during polypeptide transport along the secretory line, glycans are further modified to acquire functions such as interaction moieties for cell adhesion and recognition structures for ligands and pathogens 65-67.

Considering the polyvalent aspect of glycosylation, it is not surprising that alterations in the glycosylation efficiency cause a large spectrum of pathologies, which can be grouped in the family of the so-called congenital disorders of glycosylation (CDG) 67,68.

1.1.4 Substrate binding to malectin (MLEC)

Immediately after addition of the pre-assembled oligosaccharide to the nascent polypeptide chain, the first glucose is removed by α-glucosidase I (Fig. 1) 69-71, an ER type-I transmembrane protein 72. This di-glucosylated form has been initially considered a short- lived intermediate of protein-bound oligosaccharide processing lacking biological relevance. This view has changed with the discovery of the ER lectin malectin (MLEC), which binds to the di-glucosylated glycoproteins (Fig. 2, step 1) 73. There is evidence that MLEC is induced during ER stress 74. Moreover, it has been reported to preferentially recognize off-pathway conformers of model glycoproteins, such as the viral influenza hemagglutinin (HA) and null Hong Kong (NHK), a folding-defective variant of α-1-antitrypsin (α1AT). The ability of MLEC to identify and capture terminally misfolded proteins so early and to discriminate them from folding intermediates may depend on the formation of a functional complex with ribophorin I 74-77. Curiously, a role for MLEC in the MHC-I degradation induced by human cytomegalovirus infection has been recently proposed and proved to be ribophorin I- independent 78. It is presumable that the need for a functional complex with ribophorin I might be skipped in presence of the viral membrane protein U2 that binds to MLEC and seems to replace ribophorin I 78.

Fig. 2. Newly synthesized polypeptide’s destiny within the ER. Upon translocation and addition of the N- glycan, the first glucose of the nascent polypeptide is removed by glucosidase I. The di-glucosylated form binds to MLEC (step 1). Glucosidase II cuts the second glucose from the glycan. The resulting mono-glucosylated polypeptide interacts with CNX or CRT (step 2). Polypeptide’s release from both lectins is caused by the removal of the last glucose operated by glucosidase II (step 3). Re-glucosylation of unfolded intermediates by UGGT1 dictates the re-association with CNX or CRT (step 4). Association/dissociation cycles from CNX/CRT can take place (steps 3-4). Unfolded intermediate can attain the native structure (step 5) and leave the ER (step 6) or can terminally misfold. N-glycans displayed on terminally misfolded proteins are further trimmed (step 7) and this precedes retrotranslocation to the cytosol for proteasomal degradation (step 8).

1.1.5 Substrate binding to calnexin (CNX) and calreticulin (CRT)

α-glucosidase II cleaves the second glucose from the N-linked oligosaccharide (Fig.1). α- glucosidase II is composed by GIIα, the catalytic subunit, and GIIβ, which contains the ER retention sequence 79 and a mannose-6-phosphate receptor homology (MRH) domain, which has been suggested to control GIIα activity by associating with the mannoses of the B and C branches of the protein-bound glycan 80. This trimming step generates the mono-glucosylated form of the maturing protein that recruits the ER lectins CNX and CRT (Fig. 2, step 2) 81,82. Specialized ER-resident enzymes associated with CNX/CRT promote the oxidative folding and prolyl-isomerization of nascent proteins 83-85.

More specifically, the two lectin chaperones form functional complexes with ERp57 86, a member of the protein disulfide isomerase (PDI) family and with cyclophilin B 84, a member of the peptidyl-prolyl isomerase (PPI) family that catalyze formation of native disulfide and peptidyl-prolyl bonds, respectively (see section 2) 62. CNX and CRT are paralogues that exhibit 39% sequence homology; the main difference between the two resides in their topology. CNX is a type-I membrane protein, while CRT is an ER luminal chaperone 87. Beyond that, CNX and CRT possess similar properties, such as the presence of an N-terminal globular domain (that comprehends the lectin binding site) and a P-domain that mediates ERp57 and cyclophilin B association 84,86,88,89. Since the interaction with CNX/CRT occurs at the same region of the ER lectins 84,86, functional complexes between CNX/CRT-ERp57 and CNX/CRT-cyclophilin B seem to form separately and act sequentially during nascent polypeptides folding. Importantly, CNX and CRT are also involved in other biological processes such as calcium homeostasis, immunity, cancer and apoptosis 90,91.

1.1.6 Protein release from CNX and CRT

α-glucosidase II cleaves the third glucose (Fig. 1), thereby causing the protein release from the two lectin chaperones (Fig. 2, step 3). This is due to the different affinity that CNX and CRT display for the mono- and the de-glucosylated forms of glycoproteins 81,82,92-94. In fact, both lectins have high affinity for mono-glucosylated species and low affinity for de-

7 glucosylated forms. Folding efficiency differs from protein to protein and may require reiterated rounds of folding attempts into the CNX/CRT chaperone system 95.

1.1.7 Quality control (QC)

As a general rule, correctly folded polypeptides can leave the ER, while incompletely folded proteins are retained within the ER lumen. Protein retention is primarily mediated by the association of non-native proteins with ER-resident chaperones and folding factors, such as CNX, CRT and BiP 96, with CNX/CRT representing the best characterized route of this ER retention-based quality control 97,98.

After release from the lectin chaperones CNX and CRT (Fig. 2, step 3), the folding status of the polypeptide is scrutinized by the enzyme UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) (Fig. 1), which recognizes exposed hydrophobic patches of the substrate protein 99,100. Proteins that have reached the proper native structure pass the UGGT1-mediated ER quality control (ERQC) and are exported to their final intra- or extracellular destination. Non- native proteins are re-glucosylated by UGGT1 and re-addressed to CNX/CRT for a new folding attempt (Fig. 2, step 4) 101. Recently, UGGT2, homologue of UGGT1, has been identified and proved to possess re-glucosylation activity in vitro 102. An active role of UGGT2 in protein biogenesis remains to be proven.

Another important quality control level is represented by the thiol-mediated QC 103. This model has been hypothesized after observing that unassembled IgM subunits exposing C- terminal cysteines in B lymphocytes were not able to leave the ER. Thus, proteins displaying free thiol groups are recognized as transport-incompetent and retained in the ER. This system of ER retention involves the formation of disulfides between the transport-incompetent polypeptides and ER resident proteins, creating a proper thiol-based ER matrix 104.

1.1.8 Export of native proteins from the ER

Properly folded proteins that pass ER quality control (Fig. 2, step 5) move to the ER/Golgi intermediate compartment (ERGIC) and reach the Golgi. Recently, the existence of a novel post-ER quality control has also been reported. This checkpoint, mediated by UGGT1 and VCP/p97, prevents the transport to the Golgi of proteins with native ectodomains displaying defects at the level of the transmembrane domains 105.

Protein export from the ER occurs at the level of ER exit sites where the cargo protein is wrapped by cytosolic coatomer protein II (COPII) coated vesicles (Fig. 2, step 6) 106. Homotypic fusion events between COPII-coated vesicles generate the ERGIC compartment 107. The ER to Golgi cargo trafficking can be mediated by membrane receptors or can proceed by bulk flow 108. In the first case, the interaction cargo-receptor could be glycan- mediated or occur by protein-protein interaction 109. The removal of mannose i (Fig. 1) by ERManI 110 allows recognition of native glycoproteins by cargo receptors of the export machinery. These lectin receptors are ERGIC-53, VIPL and VIP36 111. ERGIC-53 recognizes a large spectrum of glycoproteins displaying high mannose content, thus ignoring the ERAD 111 destined Man5-6GlcNAc2 species . According to the bulk flow model, correctly folded polypeptides diffuse into COPII-coated vesicles without the need of any receptor. Protein export is negatively regulated by ER factors, in fact, unfolded/misfolded proteins that are associated with ER resident chaperones are unable to leave the ER 112.

1.2 ER-Associated Degradation (ERAD)

1.2.1 N-glycan processing: the signal for degradation

The clearance of misfolded products requires their extraction from the CNX/CRT cycle, their dislocation into the cytosol and their destruction by dedicated degradation systems. Glycosylated misfolded proteins’ degradation relies on progressive de-mannosylation of the N-glycan (Fig. 2, step 7). Mannose trimming is considered the signal for degradation of misfolded glycopolypeptides and is catalyzed by members of the glycosyl hydrolase family 47 48. The removal of mannose i by the ERManI (Fig. 1), facilitates the engagement of other

9 glycosyl hydrolase family 47 members such as ER-degradation-enhancing mannosidase-like 1 (EDEM1) 113,114, EDEM2 115,116 and EDEM3 117, which possess high affinity for misfolded glycopolypeptides and have been shown to contribute to extensive substrate de- mannosylation 114,117. ERManI is a type-II membrane protein that has been proposed to reside and concentrate in quality control vesicles, creating a compartmentalization that avoids premature mannose trimming of glycopolypeptides undergoing folding programs 118. The trimming of mannose g is particularly important as it prevents the re-glucosylation by UGGT1 and the retention of non-native glycoproteins into the CNX/CRT cycle (Figs. 1-2) 48.

Extensive de-mannosylation leading to the formation of Man5-7GlcNac2 polypeptide has also been reported 116,119 (Fig. 1).

1.2.2 Recognition of ERAD substrates

Terminally misfolded glycoproteins must be rapidly degraded to avoid the formation of toxic aggregates and ER homeostasis perturbation. Incorrectly folded polypeptides that form in the ER are eliminated through the ER-Associated Degradation (ERAD) pathway (Fig. 2, step 8), which comprehends a series of events, including recognition, preparation for dislocation, ER- to-cytosol retro-translocation and proteasomal degradation of misfolded products 120.

Specific ERAD lectins are responsible for the identification of aberrant products. Misfolded polypeptides addressed to ERAD have been subdivided in three different classes according to the position of the lesion they possess. These classes are named ERAD-L, ERAD-M and ERAD-C and comprise ERAD substrates that carry the lesion in the ER lumen, in the transmembrane or in the cytosolic portion, respectively 121. The ERAD-L group has been in turn subdivided into ERAD-Ls and ERAD-Lm, which indicate soluble and membrane proteins, respectively, that carry luminal lesions 122. Misfolded polypeptides exposing mannose j are recognized by the luminal lectins OS-9 and XTP3-B 123, which exhibit one and two MRH domains, respectively (Fig. 3) 124-126. OS-9 and XTP3-B have been found in supramolecular complexes containing HRD1, SEL1L and many other luminal, membrane- anchored and cytosolic factors, leading to hypothesize that these lectins could serve to deliver misfolded proteins to the dislocation complexes located at the ER membrane 123,125.

Specifically, the degradation of ERAD-Ls misfolded polypeptides has been shown to be mediated by a functional complex containing HRD1, SEL1L, OS-9 and XTP3-B, suggesting an overlapping function of the two lectins 122,127. In the case of non-glycosylated proteins, OS-9 and XTP3-B have been shown to cooperate with the ER chaperones BiP and GRP94 in the clearance of misfolded proteins 123,128, demonstrating that both lectins are able to bind to misfolded products aside from their glycosylation profile 127.

1.2.3 Preparation for dislocation across the ER membrane

Once the terminally misfolded products have reached the ER dislocation complex, polypeptide chains must be prepared for retro-translocation across the ER membrane into the cytosol 129,130. PDI and PPI enzymes catalyze the unfolding of polypeptide chains through disulfide bonds reduction and peptidyl-prolyl bonds isomerization, respectively 131. The PDI member ERdj5, for example, has been implicated in the reduction of NHK covalent dimers, allowing substrate’s retro-translocation and clearance 132. Interestingly, this process is thought to be assisted by the action of the ER chaperone BiP 133. Likewise, a role for PDI, ERp57, ERp72 and ERp29 in the disassembly of ternary/quaternary protein structures has been described and proved to be required for protein unfolding and degradation 134. On the same line, the PPIase activity of cyclophilin B has been shown to determine the catalysis of cis-trans peptidyl-prolyl bonds isomerization within the ERAD substrate BACE457∆ sequence, facilitating its transport to the cytosol for degradation 135. Retro-translocation of aberrant products that do not require unfolding and linearization has also been reported 136. More importantly, since entire virions exceeding the size of a dislocation channel have been reported to be able to retro-translocate in the cytosol, the requirement for linearization and unfolding to achieve dislocation as well as the identity of the putative channel for protein retrotranslocation is still under debate 137.

Fig. 3. Schematics of an ER dislocation machinery engaged by misfolded polypeptides. A HRD1-based dislocon is depicted. E3, E3 ligase; E2, E2 ligase; R, Ring finger; Ub, ubiquitin chain.

1.2.4 ER-to-cytosol polypeptide’s translocation, poly-ubiquitination and proteasomal degradation

ER dislocation machineries are large protein complexes located in the ER membrane (Fig. 3). These are designated for the ER-to-cytosol retro-translocation of soluble secretory misfolded proteins and for dislocation of integral membrane defective proteins 138. Although exceptions do exist, protein retro-translocation/dislocation is coupled to substrate’s ubiquitination, a mechanism that requires the concerted action of dedicated enzymes. In fact, ubiquitin is activated by E1 enzymes, transferred to ubiquitin-conjugating E2 enzymes and finally delivered to E3 ubiquitin ligases, which catalyze the ubiquitination of select substrates 139. ER dislocation machineries are built around ER membrane-anchored E3 ligases. Mammalian cells possess a broad spectrum of E3 ubiquitin ligases (24 members identified so far 140) that mediate ubiquitination of substrates displaying different chimico-physical properties 131. HRD1 and GP78 are the two better characterized mammalian E3 ligases, being

12 involved in the disposal of a multitude of misfolded polypeptides 122,141-143. Interestingly, while ERAD-Ls substrates’ degradation strongly depends on the HRD1-based dislocon, the disposal of several model ERAD-Lm polypeptides is mediated by the GP78-based dislocation complex 122. Dislocation machineries also contain adaptor proteins that ensure the correct substrate recognition and regulate the activity of E3 ligases 144. One of these is SEL1L, a crucial factor that is responsible for HRD1-based dislocon stabilization (Fig. 3) 145,146. Knock-down of SEL1L has been proved to prevent degradation of some ERAD-Ls substrates, without affecting degradation of several ERAD-Lm polypeptides 122,147,148. HERP is another fundamental adaptor that, having short half-life, might be required as a regulator for the assembly and stabilization of the HRD1-based dislocon (Fig. 3) 146. Among the several hypothesis that have been made, the HRD1-based dislocon is thought to be one of the putative proteinaceous channel through which terminally misfolded polypeptides are transported outside the ER 149. The requirement for a dislocation channel might be of primary need for the degradation of luminal misfolded proteins, while integral membrane misfolded products might engage different degradative mechanism, such as lipid droplets 150,151 or autophagy 152. In addition, several membrane proteins possess degradative signals, such as destabilizing charged amino acids within the transmembrane regions, which are recognized by intramembrane rhomboid proteases 153.

Polypeptide’s retro-translocation into the cytosol is driven by ubiquitin conjugation and by the ATPase activity of VCP/p97 154,155. As the polypeptide chain emerges in the cytosol, its degradation relies on the ubiquitin-proteasome system (UPS), a complex machinery that mediates targeted protein destruction 156. To avoid polypeptide aggregation within the cytosol, retro-translocation and proteasomal degradation are thought to be tightly coupled, so that degradation occurs co-translocationally 157.

1.2.5 ERAD machineries are hijacked by pathogens

In order to intoxicate cells, certain types of viruses and toxins go through the secretory pathway in a retrograde manner, entering into the ER in order to be activated prior to transport into the cytosol 158,159. ER dislocation complexes are thought to be specifically

13 hijacked for pathogens’ entry into the cell cytosol 160. Several viruses, such as non-enveloped viruses, exploit the ER to change their conformation and gain a translocation-competent form suitable for cytosolic access and infection. For the non-enveloped viruses polyomavirus (Py) and simian virus 40 (SV40), ER membrane penetration 161 is assisted by protein disulfide isomerases such as PDI, ERp72 and ERp57 162-164. These enzymes reduce and/or isomerize viral disulfides to generate membrane-transport competent forms of the viruses 160. The dislocon components SEL1L and Derlin 1 also contribute to viral penetration, together with the ER chaperone BiP and the ERAD factors BAP29 and BAP31 137,163. On the contrary, HRD1, HERP, EDEM1 and P97 seem to be dispensable for this process 165.

Certain bacterial and plant toxins, called AB toxins, need the ER environment to get activated through the reduction of an inter-chain disulfide between the catalytic (A) and the regulatory (B) subunits. Among them, the bacterial cholera toxin and the plant ricin are the better characterized 165. The retro-translocation into the cytosol of the active A subunit of both toxins relies on the intervention of HRD1, SEL1L and Derlin 2 166. While the reductase for cholera toxin’s activation remains unknown 160, ricin reduction seems to be mediated by PDI and TMX1 167 (see section 1.5.2.4.2.1).

1.3 ER stress and the unfolded protein response (UPR)

Misfolded polypeptides accumulation in the ER lumen, alterations of the ER environment and of the ER cargo load induce conditions of ER stress 168. Eukaryotic cells experience ER stress during several physiological and pathological states, i.e., cell differentiation, infections, intoxications and drug treatments. Thus, they have evolved peculiar mechanisms to face these delicate circumstances. As the ER stress reaches a certain threshold, the UPR pathway is activated. The UPR induction establishes an ER-to-nucleus communication, leading to inhibition of the global protein synthesis (i), specific production of select ER chaperones and ERAD factors (ii) and ER volume enlargement by increasing lipid biogenesis (iii) 169. Mammalian UPR is mediated by three distinct arms: protein kinase RNA-activated-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1). These key elements are ER membrane proteins responsible for the transmission of

14 stress signals to the nucleus 170. Genes that are selectively up-regulated by UPR contains ER stress responsive elements within the promoter region 171. The activation of UPR programs allows the re-establishment of the ER proteostasis. When ER stress conditions cannot be relieved, cell death is eventually induced 172.

1.4. Rate-limiting reactions during protein folding and ERAD

1.4.1 Peptidyl-prolyl bonds isomerization

Peptide bonds within polypeptide chains are planar and their flanking Cα atoms can exist in either cis or trans conformations. While peptide bonds between residues other than proline preferentially adopt trans conformation, for peptide bonds connecting any amino acid with proline (Xaa-Pro or prolyl-bond), the trans conformation is only marginally favored over the cis 173-175. The cis-trans isomerization of prolyl-bonds is a rate-determining reaction during protein folding since the spontaneous cis-trans conversion is slow as it occurs in the order of hundreds of seconds 129,176,177 if not catalyzed by enzymes belonging to the PPI family (Fig. 4) 62,176,178.

Figure 4. Mechanism of peptidyl-prolyl cis- trans isomerization catalyzed by PPI proteins (from 62).

Protein of the PPI family are categorized in four sub-groups: parvulins, cyclophilins (Cyps), FK506 binding proteins (FKBPs) and the serine/threonine-protein phosphatase 2A activator (PTPA) 62,179-181. Cyclophilins are inhibited by the immunosuppressive and anti-rejection drug cyclosporine A (CsA). FKBPs are inhibited by FK506 and by rapamycin 182-184.

About 20 putative Cyps, 15 FKBPs, 3 parvulins and 1 PTPA genes have been identified in the human genome 180. Among them, six members of the FKBPs and two members of the Cyp sub-groups are localized in the ER of mammalian cells 62,185,186. These are FKBP13, FKBP19, FKBP22, FKBP23, FKBP60, FKBP65, CypB and CypC 62,185. Although in vitro experiments have shown that these enzymes are capable to speed up folding of client polypeptides 187, evidences about their activity on protein folding in vivo remain indirect.

In addition to the PPIase activity, some FKBPs also display chaperone activity and bind exposed hydrophobic regions of misfolded polypeptides, preventing their aggregation 181. In the case of FKBP65, both the PPIase and the chaperone-like activities have been shown to mediate the appropriate folding of the proline-rich proteins collagen and tropoelastin 188.

An important contribution by CypB to the in vitro folding and assembly of IgG has been reported. This role was attributed to the formation of functional complexes between CypB and select PDI members, such as ERp72 and PDI 187,189. Analogous productive interactions between PPI and PDI foldases, such as FKBP23-ERp29, FKBP65-ERp19 and CypB-P5 have been also proposed. Curiously, CypB was also found to interact with the major ER Ca2+- binding chaperones BiP and GRP94, thus regulating ER calcium stores via its PPIase activity on both chaperones 81.

Beyond its importance during protein folding, PPIase activity is also required during misfolded proteins’ degradation (see section 1.2.3).

1.4.2 Disulfide bonds formation, isomerization and reduction

Disulfide bonds are defined as covalent linkages between two cysteine residues 190. The chemical reaction consists in the oxidation of the thiol groups of the two cysteines involved. As such, these reactions are more likely to occur in the ER, which is a more oxidizing environment than the cytosol 191, although disulfide bond can also form in the cytoplasm 192 and in mitochondria 193. Evidences from different laboratories agreed on a value of about - 225 mV as the redox poise of the ER lumen 194-196. The estimation of the ER redox potential is based on the ratio between the individual concentration of reduced and oxidized

16 glutathione (GSH/GSSG ratio). A GSH/GSSG ratio of about 3:1 is considered as the appropriate redox environment for disulfides formation. Glutathione is a low molecular weight cysteine-containing compound that is ubiquitously expressed and found to reach a concentration up to 15 mM in the ER lumen of mammalian cells 197. A recent report seem however to challenge the role of glutathione in the ER redox regulation and in protein biogenesis 198, as the total depletion of the reduced pool of glutathione did not result in the impairment of the reductive capacity of the ER.

Thiol oxidation can be catalyzed by multiple factors and pathways, such as molecular oxygen 199, low molecular weight effectors (i.e., glutathione) 200, reactive oxygen species (ROS) 201, dehydroascorbate 202 or upon intervention of dedicated enzymes belonging to the thioredoxin superfamily 203-206. This range of possibilities significantly complicates the characterization of each contribution to disulfide bond formation under physiological conditions also because inhibition of one pathway or inactivation of one enzyme activates compensatory mechanisms that maintain cellular proteostasis 207,208. Disulfide bonds can form within the same polypeptide chain (intramolecular) or between cysteines of two distinct polypeptides during oligomerization (intermolecular). As such, disulfide bonds stabilize the tertiary and/or quaternary structure of a protein 209. Since every cysteine has the potential to form a disulfide bond, the greater the number of cysteines in a polypeptide, the greater is the probability to form non-native disulfides. For this reason, disulfide bond formation is often a rate- determining reaction during protein folding and specific catalysts are in place to successfully accomplish this crucial task 210.

Figure 5. Oxidation, reduction and isomerization reactions catalyzed by PDI family members. Oxidative pathway catalyzed by PDIs (upper panel). A PDI member in the oxidized state (PDIox) forms a mixed disulfide (MD) attacking a substrate’s thiol (step 1). From the MD, there are three possibilities: the first option is that another substrate’s thiol attacks the PDI-substrate MD (step 2), resulting in the oxidation of the substrate and the PDI reduction. The second option is a nucleophilic attack by the C-terminal active site cysteine of the PDI on the MD (step 3). The last possibility is a nucleophilic attack by a substrate’s thiol on a substrate’s disulfide (step 4), resulting in an isomerization reaction. The resulting MD is subjected to the same reactions described in steps 2-4. Reductive pathway catalyzed by PDIs (lower panel). A PDI family member in the reduced state (PDIred) forms a MD through a nucleophilic attack of the N-terminal active site cysteine on a substrate’s disulfide bond (step 5). Here there are two options: the first is the nucleophilic attack of a different substrate’s thiol on the MD, resulting in an isomerization reaction (step 6). The second is the nucleophilic attack by the C-terminal active site cysteine on the MD, leading to the reduction of the substrate and PDI oxidation (step 7). Adapted from 211.

1.5 The PDI family

The mammalian ER contains 23 PDI family members (Table 1). These are characterized by the presence of one or more thioredoxin-like domains (Trx) and by ER localization 203. Given the differences in functional properties and protein organizations, it is presumable that PDIs are not equivalent and specifically work toward select subsets of substrates 83,211. However, such a great number of PDI proteins within the ER suggests a possible functional redundancy 211. This aspect is largely supported by the absence of evident phenotypes on protein secretion in individual PDI knock-out models 207,212. Understanding why so many PDIs are present and which are their specific tasks is a question that is far from being answered.

Table 1. Protein disulfide isomerase family. a, active thioredoxin-like domain; b, inactive thioredoxin-like domain; x, linker domain; J, J-domain; t, transmembrane domain. The amino acidic composition of the PDI active sites is shown in the third column. O, oxidation; R, reduction; I, isomerization; C, chaperone activity (from 62).

1.5.1 The structure of PDI

Domain architecture changes significantly between PDIs. The “classical” organization is the one displayed for example by PDI, the first family member that has been reported 213. PDI possesses two catalytic domains, a and a’, and two non-catalytic domains b and b’. b’ and a’ are separated by a linker region of 19 amino acids that is called x domain 205,214, resulting in the abb’xa’ domain organization (from the N- to the C-terminus). While the exact role of the b domain is still unknown, the b’ domain binds substrates via hydrophobic interactions 215. Interestingly, it has been shown that while the non-catalytic b’ domain is able to bind to small peptides, a and a’ domains contribute to the binding to larger non-native peptides. The structural information about PDI derives from NMR and crystal structures of the human PDI a, b, a’, b’x and bb’ protein segments 216-219. To date, the crystal structure of the full-length PDI protein has not been solved, but crystals of several other PDI members, such as ERp57, ERp44, ERp29 and the yeast Pdip have been defined 220-223. As reported in Table 1, PDIs exhibit different domain combinations, with ERp57, PDIp and PDILT displaying the same architecture of PDI, and ERdj5 and the membrane-bound family members being structurally distinct because of the presence of additional J and t domains, respectively.

PDI catalytic domains contain Cys-Xxx-Xxx-Cys active sites that during the catalytic cycle exist in di-thiol, disulfide and mixed disulfide states (Fig. 5) 205. Several PDIs do not contain active sites or do not have classical CXXC active sites, meaning that not all the members are functional oxidoreductases (Table 1). The first case regards ERp27 and ERp29, which do not have a or a’ domains and only display non-catalytic thioredoxin-like domains. For this reason, the exact contribution of ERp27 and ERp29 to protein biogenesis in the ER remains elusive. In the second case, alternative CXXC active sites lacking the N-terminal, the C- terminal or both the N- and the C-terminal cysteines do exist. Enzymes lacking the N- terminal or both active site cysteines, such as TMX2 and PDILT, are unable to perform nucleophilic attacks and are thus inactive. For PDIs lacking the C-terminal cysteine instead, as in the case of ERp44, AGR2, AGR3 and TMX5, the N-terminal active site cysteine is able to perform a nucleophilic attack and has the potential to establish mixed disulfides (MD) with substrates. The limitation here is that the C-terminal cysteine is not in place to resolve the MD, so that the reaction can only be concluded with the help of “external” thiols provided by

20 the substrate itself or by other PDI members. ERp44 is the best characterized PDI member lacking the C-terminal active site’s cysteine. The presence of the single N-terminal cysteine allows to ERp44 to establish mixed disulfides with the redox partner Ero1-Lα and with substrates, such as Ig-K and Ig-J chains. Moreover, it confers to ERp44 the ability to bind to exposed thiol groups during thiol-mediated quality control 224,225.

For canonical PDI containing CXXC active sites, the identity of the two residues X differs from protein to protein (Table 1) and plays an important role in determining the reduction potential of each enzyme 205. In the case of PDI, the active site sequence is CGHC for both a and a’ domain. The histidine residue is thought to decrease the pKa of the N-terminal cysteine 226, stabilizing the disulfide state of the enzyme. Instead, for PDIs that contain proline residues between the active site cysteines (i.e., ERdj5, TMX1, TMX4), the disulfide state is destabilized by steric hindrance, so that the active site di-thiol form is favored 205,227. PDIs with active sites in disulfide state (or oxidized state) are supposed to behave as oxidases, while enzymes with active sites in di-thiol form (or reduced form), work prominently as reductases 205.

Many PDI family members contain in the promoter region ER stress element (ERSE) motifs, which are responsible for gene induction upon ER stress. This way of regulation underlines an important role for PDIs during the UPR (see section 1.3) 206.

1.5.2 Select examples of PDI proteins

1.5.2.1 PDI

PDI is the first family member that has been identified 213. It is ubiquitously expressed throughout tissues and organs and considered as one of the most abundant proteins of the ER 228. Human PDI is a protein of 508 amino acids that contains an N-terminal signal sequence, four Trx-domains and a C-terminal ER retrieval sequence. Beyond its oxidoreductase activity, PDI also displays chaperone-like activity 229. PDI importance is underlined by the non-viability of its constitutive depletion 230. It is only marginally induced upon ER stress, probably because of its abundance 206. In the contest of protein production, PDI has been

21 reported to be required for the oxidative folding of MHC-I heavy chain, thyroglobulin, Semliki forest virus glycoproteins, immunoglobulins and interferon-gamma 231-235. In line with a role for PDI during protein folding, a putative association of PDI with the P-domain of CRT has been proposed 236. However, follow-up studies revealed that this interaction could only take place under non-physiologic ER calcium concentration, excluding the idea that PDI could work in association with CNX/CRT 62. In vitro analysis of antibody chains oxidative folding demonstrated a cooperation between PDI and BiP. It has been proposed that the cysteine-pairing activity of PDI is facilitated by BiP, which binds to unfolded polypeptides, exposing the otherwise buried free cysteines of the client protein 237.

1.5.2.2 ERp57

ERp57 is the PDI member that most resembles the domain architecture of PDI. Like PDI, the 505 amino acids-long ERp57 displays an N-terminal signal sequence, four Trx-domains and a C-terminal ER retrieval sequence 206. The main difference between PDI and ERp57 lies in the b’ domain, which confers chaperone-like activity to PDI. The ERp57 b’ domain lacks the hydrophobic patch of PDI, contains instead positively charged amino acids 238,239 and mediates association with the P-domain of both CNX and CRT. This confers to ERp57 the function of master oxidoreductase during glycoprotein maturation 240,241. Consistently, the efficiency of glycoprotein oxidative folding in vitro is hampered when ERp57 association with CNX/CRT is impeded 242. Several ERp57 clients, such as MHC-I heavy chain, influenza hemagglutinin, clusterin, β1 integrin and progranulin have been identified 212,243-245. Unexpectedly, ERp57 knockout cells do not have obvious redox alterations: this evidence suggests that other PDI members compensate ERp57 loss. In agreement with that ERp72 has been shown to substitute ERp57 212.

1.5.2.3 ERdj5

ERdj5 is a peculiar member of the PDI family that also belongs to a second protein family, the ERdj family, which comprises co-factors that stimulate Hsp proteins’ ATPase activity 246.

ERdj5 is 793 amino acid long and possesses an N-terminal signal sequence, a J-domain, five Trx-domains and a C-terminal ER retrieval sequence 206. ERdj5 is induced by ER stress. ERdj5 knockout mice have been generated and found to have a very weak phenotype. Indeed, ER stress has been observed exclusively at the level of the salivary gland 247. The C- terminal region of ERdj5 contains the site for the interaction with EDEM1: importantly, this C-terminal domain lacks N-glycosylation sites, proving that the association between ERdj5 and EDEM1 is glycan independent 133. The J-domain of ERdj5, instead, mediates the association with the ER chaperone BiP 248. According to the literature, the major role for ERdj5 is in the ERAD pathway and depends on its association with EDEM1 and BiP 132,133,249. The model of the ERdj5-mediated ERAD pathway that has been proposed implies that a complex formed between EDEM1 and ERdj5 recognizes NHK aberrant dimers, allowing substrates’ reduction. Afterwards, BiP associates with the complex and mediates the transfer of reduced NHK molecules to the retro-translocation channel 133.

More recently, a role for ERdj5 in protein folding has been proposed. Studies on the low- density lipoprotein receptor (LDLR), known to form non-native disulfides during its maturation, demonstrated that ERdj5 is required for the formation of the correct set of disulfides of LDLR 250. Importantly, the association between ERdj5 and BiP appeared to be required for LDLR proper folding.

1.5.2.4 Membrane-anchored PDI

1.5.2.4.1 MagT1 and Tusc3

Most of the PDI family member are soluble proteins. However, 7 of them are anchored at the ER membrane. These are the two OST complex subunits MagT1 and Tusc3 and the five members of the thioredoxin-related transmembrane group TMX1-5.

Recent studies 52,251, characterized the function of the MagT1/Tusc3 subunits of the OST complex. As for the catalytic subunits STT3A and STT3B, MagT1 and Tusc3 are mutually exclusive within the OST. In particular, STT3B and MagT1 belong to the same OST complex type 251. Both MagT1 and Tusc3 are multi-pass membrane-bound proteins, which

23 possess oxidoreductase activity, displaying peculiar CXXC active site motifs (CVVC for MagT1 and CSVC for Tusc3). MagT1 and Tusc3 are paralogues and share 70% sequence identity. They are the orthologs of yeast Ost6p and Ost3p, respectively 60. Their main function is to facilitate and increase N-glycosylation efficiency by establishing transient mixed disulfides with newly synthesized glycoproteins 52,53,251, decreasing polypeptides’ folding rate.

1.5.2.4.2 The thioredoxin-related transmembrane (TMX) subgroup

1.5.2.4.2.1 TMX1 Thioredoxin-related transmembrane protein 1 (TMX1) has been identified in 2001 252. TMX1 is a non-glycosylated ER type-I membrane-protein of 281 amino acids. It has an N-terminal signal sequence but lacks a canonical C-terminal ER retention sequence 253 and displays instead a cytosolic di-arginine RQR motif, which has been proposed to confer to TMX1 the ER localization 254. TMX1’s sub-organellar localization is post-translationally regulated, moving between the rER and the MAM subdomains upon palmitoylation on two cytosolic cysteine residues 255, suggesting that TMX1 might have a role at the MAM in lipid transfer and calcium homeostasis. TMX1 contains a single luminal a domain with a peculiar CPAC active site. TMX1 has reducing activity, catalyzes refolding of scrambled RNase and can reduce insulin disulfides in vitro 252,256. Consistent with the lack of an ERSE motif in its promoter, TMX1 is not induced upon ER stress and its knockdown in mammalian cell cultures does not lead to UPR activation 257. TMX1 interacts with CNX but not with the luminal paralogue CRT. Consistently, the association is mediated by the respective transmembrane domains 257. The expression of TMX1 is ubiquitous, reaching high levels in liver, kidney, placenta and lungs. Recently, the TMX1 knockout mouse has been generated and found to have no evident defects. Consistent with the high expression level in the liver, the TMX1 knockout mice exhibit susceptibility to liver inflammation induced by lipopolysaccharide and D-galactosamine treatments 258. An important interactor for TMX1 is VKOR, a well-known factor of the ER electron transfer pathway. Being VKOR also a membrane-bound protein highly expressed in the liver, it has been postulated that it could

24 represent the redox partner for TMX1 259. Until recently, functional evidences about TMX1 were quite weak. The principal functional implication for TMX1 regarded its ability to form mixed disulfides with MHC-I heavy chain, as already shown for PDI and ERp57 257. Moreover, a role of TMX1 in the ERAD process has been hypothesized due to its involvement in AB toxins ricin and abrin reduction for translocation across the ER membrane 167. The TMX1-catalyzed reduction of ricin/abrin suggests that TMX1 is putatively part of an ER functional complexes that are hijacked for cell intoxication.

We think that the work presented in this thesis contributed to a better functional characterization of TMX1, regarding its role in protein biogenesis and degradation.

1.5.2.4.2.2 The other TMX proteins Beyond TMX1, other four proteins belong to the TMX sub-group (Fig. 5). TMX2 has been discovered in 2003 and is still poorly characterized 260. It is a protein of 296 amino acids with an N-terminal signal sequence, a single a domain with the SNDC active site, a single transmembrane domain and a C-terminal KKXX-like retention motif. Curiously, lacking the N-terminal active site cysteine, TMX2 is enzymatically inactive. Moreover, unlike the other TMXs, TMX2 seems to have a type-II orientation, having the inactive SNDC motif in the cytosol rather than in the ER. To date, experimental evidences on TMX2 functional role in vivo are missing.

Figure 5. TMX sub-group of the protein disulfide isomerase family.

TMX3 has been discovered in 2005 261. It is a 454 amino acids long type-I membrane protein displaying an N-terminal signal sequence and an abb’t domain architecture comprising a CGHC active site. TMX3 possesses two N-glycosylation sites in the luminal portion and a canonical ER retention sequence KKKD in its cytosolic domain. Importantly, the CHGC composition of the active site is the most spread catalytic active site’s sequence of PDI family members 203. Initial studies on TMX3 oxidoreductase activity showed that it has oxidase activity in vitro. In the same study, the ER stress unresponsiveness of TMX3 has been proved 261.

TMX4 is the paralogue of TMX1 and has been discovered in 2010 262. It is a type-I membrane protein of 349 amino acids with a single N-glycosylation site. TMX4 has an N- terminal signal sequence, a single a domain with the CPSC active site. As TMX1, TMX4 lacks a canonical ER retention sequence and possesses instead the RQR motif, a di-arginine motif that has been proved to confer ER localization 254. Moreover, as TMX1, it does not contain ERSE elements in the promoter and it is not up-regulated by ER stress. The active sites of TMX1 and TMX4 have in common the presence of an inner proline residue, which has been shown to destabilize the disulfide state and favor the di-thiol form of the active sites 205,227. Consistently, both TMX1 and TMX4 display reductase activity in vitro 257,262. A functional analysis of TMX4 revealed that it interacts with CNX and ERp57, thus a role for TMX4 in oxidative folding has been hypothesized 262. Moreover, a possible role as ERAD reductase has been investigated by looking at NHK degradation’s rate in TMX4 knockdown cells. In these experimental conditions, NHK degradation was unaffected, suggesting that TMX4 might not be required for NHK dimers reduction or that other PDIs compensate the loss of TMX4 262.

For TMX5 there are no published evidences to date. TMX5 is a predicted type-I membrane protein with an N-terminal signal sequence, a single a domain displaying the CRFS active site, four potential N-glycosylation sites and no ER retention sequence. Notably, the active site lacks the C-terminal cysteine, conferring to the enzyme the feature of natural trapping PDI member.

1.6 Mechanisms of action of PDI proteins

Apart from the two residues contained in the CXXC active sites, many other aspects (i.e., pH, redox conditions, a conserved arginine residue within the PDIs amino acidic sequence) are known to modulate PDIs activity, so that the active site composition can only be partially informative on the type of reaction (oxidation vs. reduction vs. isomerization) preferentially catalyzed by a select PDI protein 205,263. The redox state of active sites (di-thiol, disulfide or mixed disulfide) has important functional implications. Enzymes with active sites in di-thiol form, the so-called reduced form, might work as reductases (Fig. 5, step 5). Instead, PDIs with active sites in disulfide state (or oxidized state) might rather behave as oxidases (Fig. 5, 264-266 step 1) . In line with this, for PDIs with a low N-terminal cysteine pKa, the disulfide state is favored, conferring to these enzymes the ability to catalyze oxidation reactions. Due to the fact that during the catalytic cycle PDIs pass from the oxidized to the di-thiol state or vice versa (Fig. 5), cellular processes that re-oxidize or re-reduce PDI proteins must exist. Many oxidative pathways have been characterized and rely on ER oxidoreductin 1 (Ero1), peroxiredoxin IV (PrxIV), glutathione peroxidase and vitamin K epoxide reductase (VKOR) 267-271. Importantly, enzyme-catalyzed reductive processes have not been identified yet 272. It has been recently proposed that disulfide exchange reactions between individual PDI members might occur 272.

Published evidences on the link between oxidoreductase activity and the domain composition of human PDI estimated that thiol-disulfide exchange may be simply catalyzed by a or a’ domain of the enzyme, that simple isomerization requires either ab’ or a’b’ domain organization while complex isomerization instead requires the full-length protein 273,274.

The comprehension of the specificity exhibited by individual PDIs and the dissection of the exact functional role they are destined for is complicated by the fact that knock-out or knockdown of select PDI proteins often do not lead to phenotypes, possibly because of the high degree of redundancy among PDIs 83,207.

The extremely short-living nature of the inter-molecular MD formed between the oxidoreductase and the substrate during the catalytic cycle hampers the identification of PDI- substrate interactions. Chemical crosslinkers have been used to stabilize PDI-substrate

27 interactions in vivo 211, although these compounds can easily originate artefactual results. Another option is the employment of cell-permeable alkylating agents, blocking thiol- disulfide exchanges. This allowed for example the identification of MD between ERp57 and Semliki forest virus glycoproteins 233 and between PDI and the ectopically expressed folding- defective glycoprotein BACE457 130. However, the proportion of endogenous ERp57/PDI in MD-linked complex with the substrate turned out to be extremely low. For this reason, in order to accomplish a more informative analysis, an increase in the proportion of MD-linked PDI-substrate complexes should be achieved. This could be done by making use of specific constructs encoding for the so-called “trapping mutant” versions of PDIs.

Two possible trapping mutant versions of PDIs do exist: the first involves the mutation to alanine or to serine of the C-terminal active site cysteine of the CXXC motifs and has been used, for example, to identify ERp57 endogenous substrates 244. The second involves the mutation of the conserved cis-proline in the thioredoxin fold by threonine (so-called proline trapping mutants). This trapping mutant version is commonly used to study prokaryotic oxidoreductases and has been employed for the first time to identify E.coli DsbA endogenous substrates 275. The main difference between the two trapping mutants resides in their ability to trap different reactions during the catalytic cycle. In fact, since the active site cysteines forms a disulfide when the enzyme is the oxidized state (Fig. 5), the replacement of the C- terminal cysteine to serine or alanine abolishes the oxidation of the enzyme, precluding the trapping of reactions on the oxidative pathways (i.e., direct oxidation and isomerization starting from the oxidized state, Fig. 5, steps 2-4). Thus, C-terminal active site trapping mutants are uniquely able to trap substrates on the reductive pathway 211. In contrast, the proline trapping mutants do not display alterations at the level of the active sites, being suitable for the identification of substrates on both the oxidative and the reductive pathways.

2. Results

2.1 TMX1, an ER stress non-responsive ER-resident member of the PDI superfamily

TMX1 is a member of the PDI superfamily associated to the ER membrane that is highly expressed in kidney, liver, placenta and lung 252. The presence of a proline residue at position 2 of the TMX1 catalytic site (CPAC, residues 56-59; Fig 1A) hints at a possible role of TMX1 as an ER reductase 227. Deletion of the TMX1 gene results in susceptibility to liver damage in mice, leading to cellular oxidative stress and hepatic injury upon treatment with lipopolysaccharide and D-(+)-galactosamine 258.

Several ER-resident oxidoreductases such as PDI, ERp44, ERp57, ERp72, ERdj5 and P5 are up-regulated upon induction of an ER stress 171,206,224,248,276-278. To determine whether TMX1 is a stress-induced member of the PDI superfamily, we exposed human embryonic kidney (HEK293) cells for 5 hr to tunicamycin. Tunicamycin is an inhibitor of N-glycosylation, commonly used in biochemistry as an ER stress-inducing agent 169. Analysis of cellular transcripts revealed that in contrast to BiP used as positive control for ER stress induction, the level of TMX1 transcripts in mock-treated and in tunicamycin-treated cells did not change (Fig. 1B). These results reveal that TMX1 levels are not regulated by the UPR, consistent with the fact that the promoter region of TMX1 lacks ER-stress responsive elements (ERSE motifs) 206.

2.2 TMX1 in vivo study: TMX1C/A trapping mutant

During the reaction cycles leading to disulfide bond formation, disassembly or rearrangement, active site cysteines of PDIs transiently form short-lived mixed disulfides with surface-exposed cysteine residues of proteins expressed in the ER lumen 233,279. The replacement of the resolving carboxy-terminal cysteine residue in the PDIs catalytic site efficiently traps mixed disulfides in the reductive pathway (Fig. 1C) 205. Thus, PDI trapping mutants have been used to identify endogenous substrates of select ER-resident oxidoreductases such as ERp57, PDI, P5, ERp18, ERp72, ERp46 and ERdj5 83,244,250,259,280.

Here, we generated plasmids encoding for the wild-type (TMX1; Fig. 1A) and the trapping mutant version of TMX1 (TMX1C/A; Fig. 1A).

2.3 Ectopically expressed TMX1 and TMX1C/A are ER-resident proteins

To determine the intracellular localization of ectopically expressed TMX1 and TMX1C/A, we performed an immunofluorescence analysis as described in the Methods section. As shown in

Fig. 1D, TMX1 and TMX1C/A are ER-resident proteins as indicates by the co-localization with the conventional ER marker CNX (Fig. 1D, Merge).

Figure 1. TMX1 wild-type and TMX1 trapping mutant: sequence, regulation, subcellular localization. A, TMX1 and TMX1C/A constructs. SS, signal sequence, Trx, thioredoxin-like domain, TM, transmembrane domain. The sequence of the active site of TMX1 and TMX1C/A and the position of the active site residues are shown. B, Human embryonic kidney (HEK293) cells were treated for 5 hr with the ER stress inducer tunicamycin. qPCR on TMX1 mRNA was performed in mock-treated and in tunicamycin- treated cells. BiP was used as positive control for ER stress induction. C, Mechanism of reduction of disulfide bonds catalyzed by members of the PDI family. The formation of a mixed disulfide by the nucleophilic attack of the N-terminal active site cysteine on a substrate disulfide bond is shown. The resulting mixed disulfide can be resolved upon nucleophilic attack of the enzyme C-terminal active site cysteine on the mixed disulfide. The replacement of this resolving cysteine (amino acid 59 in the case of the TMX1 sequence) with an alanine residue substantially stabilizes mixed disulfides between the enzyme and its substrates. D, HEK293 cells were transfected with TMX1 and TMX1C/A encoding plasmids. Cells were stained with anti-CNX and anti- TMX1 antibodies and analyzed by immunofluorescence microscopy. Scale bar:10 μM.

2.4 TMX1C/A discriminates between substrates’ topologies

2.4.1 TMX1C/A preferentially associates with membrane-bound substrates: evidences from mass spectrometry analysis

To identify endogenous substrates of TMX1, we expressed the trapping mutant TMX1C/A (Fig. 1A) in mouse embryonic fibroblasts (MEFs) and performed a mass spectrometry analysis of the cellular proteins co-immunoisolated with the ectopically expressed reductase. In contrast to the analyses performed for other PDIs that revealed both soluble and 83,244,250,259,280 membrane-bound proteins as endogenous substrates , TMX1C/A selectively trapped a series of cysteine-containing membrane proteins (Table 1).

Gene Protein names Entry Luminal Protein topology names no. Cysteines

Itgb1 Integrin beta-1 P09055 56 Single-pass type I

Slc3a2 4F2 cell-surface antigen heavy P10852 2 Single-pass type II chain Ece1 Endothelin-converting enzyme 1 Q4PZA2 12 Single-pass type II

Lrrc59 Leucine-rich repeat-containing Q922Q8 2 Single-pass type II protein 59 Ncstn Nicastrin P57716 11 Single-pass type I

Atp6ap1 V-type proton ATPase subunit S1 Q9R1Q9 2 Single-pass type I

Tspan3 Tetraspanin-3 Q9QY33 6 Multi-pass

Lrp10 Low-density lipoprotein Q7TQH7 30 Single-pass type I receptor-related protein 10 Pld4 Phospholipase D4 Q8BG07 5 Single-pass type II

Table 1. Endogenous substrates of TMX1 identified by MS analysis.

2.4.2 TMX1C/A preferentially associates with membrane-bound substrates: evidences from biochemical analysis

To determine whether the selective immunoisolation of membrane proteins was symptomatic of the intrinsic specificity of TMX1 for membrane-bound substrates, we made use of a series of model polypeptides characterized by the presence of a cysteine-containing ectodomain tethered or non-tethered at the ER membrane (Fig. 2A). Firstly, MEFs were mock-transfected (EV for empty vector, Fig. 2B, lane 1), transfected with a plasmid for expression of the trapping mutant TMX1C/A (lane 2), or with plasmids for expression of TMX1C/A and the folding-competent membrane-bound BACE501 (lane 3), the folding-competent and soluble BACE501Δ (lane 4) 281, the folding-defective membrane- bound BACE457 (lane 5), or the folding-defective soluble BACE457Δ (lane 6) 130. Cells were detergent-solubilized and the ectopically expressed, HA-tagged BACE variants were immunoisolated from post-nuclear supernatants. The immunocomplexes were separated in SDS-PAGE and transferred to PVDF membranes. The presence of the BACE variants was revealed by western blot (WB) with anti-HA antibodies (Fig. 2B, upper panel). The association of TMX1C/A was assessed, in the same PVDF membrane, upon decoration of the membrane with anti-TMX1 antibodies (Fig. 2B, lower panel). The membrane-bound BACE501 is separated in two forms (G is the Golgi, mature, EndoH- resistant form and E is the ER, immature, EndoH-sensitive form of the protein, Fig. 2B, lane 3) 281. In the cell lysate, the soluble BACE501Δ is only present in the E form as the G form is rapidly released in the extracellular medium 281. BACE457 and BACE457Δ are only present in their E form since they are ER-retained, folding-defective polypeptides 130. De- glycosylated forms of BACE501Δ and BACE457Δ (D; Fig. 2B) are also visible on the membrane.

The experiment reveals that TMX1C/A associates with the membrane-bound versions of BACE (i.e., BACE501 and BACE457, Fig. 2B, lower panel, lanes 3 and 5). In contrast,

TMX1C/A is not found in immunocomplexes containing the soluble variants of BACE (i.e., BACE501Δ and BACE457Δ, Fig. 2B, lower panel, lanes 4 and 6, respectively). The selectivity of TMX1 for membrane-bound polypeptides was confirmed upon immunoisolation from the cell lysates of the ectopically expressed TMX1C/A (Fig. 2C, lower panel) that shows the abundant co-precipitation of BACE501 and BACE457 (upper panel,

32 lanes 3 and 5). Confirming the specificity of this essay, of the G and the E forms of

BACE501, only the latter, which is in the ER, is in the TMX1C/A-containing immunocomplexes (Fig. 2C, lane 3). The association with both BACE501 and BACE457 shows that TMX1 does not discriminate between folding-competent and folding-defective polypeptides.

Figure 2. TMX1C/A preferentially associates with membrane-bound substrates A, Schematics of the model polypeptides used in panels B-E. B, MEFs transfected with empty vectors (EV, lane 1), β1- tagged TMX1C/A (lane 2) or TMX1C/A in combination with HA- tagged BACE501, BACE501Δ, BACE457 or BACE457Δ (lanes 3- 6, respectively). The HA-tagged model substrates were immunoisolated from cell lysates. The upper panel shows a WB with an anti-HA antibody to reveal the model substrates, the lower panel a WB with a TMX1-specific antibody to reveal TMX1C/A. C, Same as B but TMX1C/A was immunoisolated from the same cell lysate with an anti-β1 antibody to verify the presence of the model proteins (upper panel) in the immunocomplexes. D, Same as panels B-C for the soluble HA-tagged A1AT and the membrane-anchored A1AT-BACE (lanes 1 and 2, respectively). E, Same as panels D for the soluble HA-tagged A1AT and the membrane-anchored A1AT- CD3δ (lanes 1 and 2, respectively). G, mature Golgi form; E, immature ER form; D, de-glycosylated form; *, antibody heavy chain recognized by the secondary antibody in WB.

To further confirm the preference of TMX1 for association with membrane-bound proteins, we assessed association of the TMX1 trapping mutant with the soluble protein A1AT (Fig. 2D and 282) and of two variants of A1AT that were artificially tethered at the ER membrane with two different membrane anchors (Fig. 2D, A1AT-BACE and A1AT-CD3δ). Consistent with a preferential association of TMX1 with membrane-bound polypeptides, TMX1C/A associated with A1AT only when tethered at the membrane (Figs. 2D and 2E, lanes 1 vs. 2). Interestingly, and in contrast with the case of BACE proteins, the A1AT ectodomain contains a single cysteine residue. Hence, there are no intramolecular disulfides to be attacked by TMX1. However, there is published evidence that the A1AT cysteine undergoes various reversible modifications such as S-nitrosylation, S-glutathionylation, sulfenic acid formation, S-cysteinylation and oxidation, which induce polymerization on the A1AT cysteine 283-288. Each of these modifications could be attacked by the substrate-trapping mutant of TMX1 to form a mixed disulfide.

2.5 Role of TMX1C/A in protein folding

2.5.1 TMX1C/A establishes mixed disulfides with newly synthesized membrane-bound BACE501

An active involvement of TMX1 in determining the fate of the associating proteins should involve formation of mixed disulfides as reaction intermediates (Fig. 1C). To directly assess this, we performed a pulse and chase experiment. MEFs were transfected with two empty vectors (Figs. 3A-3D, EV, lane 1), with expression vectors for BACE501, BACE501Δ,

TMX1 or TMX1C/A and an empty vector (lanes 2-5), or with expression vectors for

BACE501 and TMX1 (lane 6), BACE501 and TMX1C/A (lane 7), BACE501 and TMX1 35 (lane 8) or BACE501 and TMX1C/A (lane 9). Transfected cells were pulsed with S- methionine and cysteine for 13 min and chased for 10 min. At this time of chase, the newly synthesized BACE501 variants are still folding in the ER as confirmed by the EndoH- sensitivity of their oligosaccharides (Fig. 3E) 281. BACE (Figs. 3A-3B) or TMX1 variants (Figs. 3C-3D) were immunoisolated from detergent extracts and complexes were analyzed in non-reducing (Figs. 3A and 3C) and reducing SDS-PAGE (Figs. 3B and 3D).

In non-reducing gels, BACE501 and BACE501 expressed alone (Fig. 3A, lanes 2 and 3) or in combination with TMX1 (lanes 6-9) are separated in a series of radiolabeled bands corresponding to various oxidation forms (Figs. 3A and Fig. 3F, fully oxidized, fOx; partially oxidized, pOx, for BACE501 and BACE501 respectively). The bands with faster electrophoretic mobility correspond to BACE forms with lower hydrodynamic radius due to the presence of intramolecular disulfide bonds (fOx in Fig. 3A and Fig. 3F) that relapse into the reduced (Red) BACE501 or BACE501 forms in the reducing gel (Figs. 3B, 3D and Fig. 3F) 289. In cells where the membrane-bound BACE501 was co-transfected with the trapping mutant

TMX1C/A, separation of the BACE immunoisolates under non-reducing conditions revealed an abundant labeled polypeptide band with an apparent MW of about 90 kDa (MD, Fig. 3A, lane 7). Sample reduction dissociated the radiolabeled 90 kDa polypeptide in its components, i.e., BACE501 with an apparent MW of about 60 kDa and a radiolabeled polypeptide with an apparent MW of about 35 kDa (Fig. 3B, lane 7). Both the 90 kDa polypeptide (Fig. 3A, lane 7) and the 35 kDa polypeptide (TMX1, Fig. 3B, lane 7) are much less abundant when BACE501 is co-transfected with the wild type form of TMX1, which is unable to stabilize the mixed disulfide with the substrate. This led us to conclude that the 90 kDa polypeptide is a mixed disulfide (MD, Fig. 3A, lane 7) containing BACE501 and TMX1C/A, which is dissociated under reducing conditions releasing the radiolabeled TMX1 polypeptide of 35 kDa (Fig. 3B, lane 7). Consistent with this hypothesis, mixed disulfides were significantly more abundant when the BACE501 was co-expressed with the trapping mutant compared to the wild type version of TMX1 (Fig. 3C, lanes 7 vs. 6). Moreover, both the MD in the non- reducing gel (Fig. 3A, lane 9) and the 35 kDa polypeptide in the reducing gel were virtually absent when the trapping mutant of TMX1 was co-transfected with the soluble BACE501 (Fig. 3B, lane 9) that does not associate with TMX1 (Figs. 2B-2C). Again, MD were separated in their BACE501 and TMX1 constituents in the reducing gel (Fig. 3D, lane 7). This confirms that the TMX1 trapping mutant stabilizes the otherwise short-lived MD intermediate of the BACE501 redox reaction. Cells expressing TMX1C/A and BACE501, also contained larger Disulfide Bonded Complexes (DBC) of more than 200 kDa (Figs. 3A and 3C, lanes 7).

Figure 3. TMX1C/A selectively establishes mixed disulfides with membrane- bound BACE501. A, MEFs were transfected with empty vector (EV), BACE501, BACE501Δ, HA-tagged TMX1 or TMX1C/A (lanes 1- 5), BACE501 in combination with HA-tagged TMX1 or TMX1C/A (lanes 6-7), BACE501Δ in combination with HA-tagged TMX1 or 35 TMX1C/A (lanes 8-9). S- methionine and -cysteine radiolabeled model substrates were immunoisolated from cell lysates with anti-BACE antibodies. The immunocomplexes were separated under non-reducing conditions. B, Same as A but immunocomplexes were separated under reducing conditions. C, Same as A for complexes immunoisolated with anti-HA. D, Same as C, analysis under reducing conditions. pOx, partially oxidized BACE; fOx, fully oxidized BACE; Red, reduced BACE; DBC, disulfide-bonded complexes; MD, mixed disulfides; *, co- precipitating band at 97 kDa which corresponds to CNX. E, EndoH assay on BACE501 after 10 min chase. MEFs were co-transfected with BACE501 and an empty vector (EV, lanes 1-2), TMX1 (lanes 3-4) or TMX1C/A (lanes 5-6). Transfected cells were pulsed with 35S-methionine and cysteine for 13 min and chased for 10 min. At this time of chase, the newly synthesized BACE501 variants are still folding in the ER as confirmed by the EndoH-sensitivity of their oligosaccharides. F, Electrophoretic mobility of BACE501 oxidized and reduced forms. MEFs were transfected with BACE501. 35S-methionine and -cysteine radiolabeled model substrate was immunoisolated from cell lysates with anti-BACE antibodies. The immunocomplexes were separated under non-reducing (lane 1) and reducing (lane 2) conditions. pOx, partially oxidized BACE501; fOx, fully oxidized BACE501; Red, reduced BACE501.

Reduction of the functional complexes containing BACE501 and TMX1C/A revealed a radiolabeled polypeptide with an apparent MW of 97 kDa in Figs. 3B and 3D, lanes 7. This polypeptide was identified by WB and by specific immunoprecipitation as the lectin chaperone CNX. The increased presence of CNX in the BACE and in the TMX1C/A immunoisolates from cells over-expressing BACE501 (lane 7 in Figs 3B and 3D, respectively) compared to cells that are not over-expressing BACE501 (lanes 5 and 9), lead us to propose that CNX is a component of TMX1 functional complexes, which are assembled or stabilized in the presence of TMX1 substrates.

2.5.2 Client-mediated association between TMX1 and CNX

To assess whether CNX forms functional complexes with TMX1, we examined the consequences of cell exposure to castanospermine (CST), a glucose analog that prevents association of newly synthesized proteins with CNX 81. MEFs were mock-transfected (EV,

Figs. 4A-4B, lanes 1-2), co-transfected with EV and a plasmid for expression of TMX1C/A (lanes 3-4), co-transfected with EV and a plasmid for expression of BACE501 (lanes 5-6) or with plasmids for expression of TMX1C/A and BACE501 (lanes 7-8). Before solubilization and immunoisolation of ectopic BACE501 (Fig. 4A) or of endogenous CNX with the associated polypeptides (Fig. 4B), cells were incubated for 10h in absence (-) or in presence (+) of CST to clear the CNX chaperone system from endogenous substrates (Figs. 4A-4B, lanes 2 and 4) or from endogenous substrates and ectopically expressed BACE501 (lanes 6 and 8). In the absence of CST, CNX strongly interacted with TMX1C/A, which is a non- glycosylated protein (Fig. 4B, lower panel, lanes 3 and 7).

Figure 4. Client-mediated association between TMX1 and CNX. A, MEFs were cotransfected with an empty vector (EV, lanes 1 and 2), an empty vector and HA-tagged TMX1C/A (3 and 4), an empty vector and BACE501 (5 and 6), or BACE501 and HA-tagged TMX1C/A (7 and 8). Cells were incubated for 10 h in the absence (–) or presence (+) of CST (1 mM). The expression level of BACE501 was checked upon WB of the immunoisolated ectopic protein. B, Same as A but endogenous CNX with associated proteins was immunoisolated from cell lysates. Immunocomplexes were analyzed under reducing conditions. Ectopically expressed BACE501 and TMX1C/A were revealed with an anti-BACE and an anti-HA antibody, respectively. C, Same as A in cells treated with CST, cycloheximide (Chx), or tunicamycin (Tun) for 3 h. D, Same as B for cells treated with CST, Chx, or Tun. G, mature Golgi form of BACE501; E, immature ER form; D, de-glycosylated form.

This association was substantially reduced upon CST treatment (lower panel, lanes 4 and 8; TMX1 is not glycosylated, thus excluding the possibility of its direct, glycan-lectin association with CNX). Thus, inhibition of endogenous (Fig. 4B, lane 4) and endogenous + ectopic (lane 8) substrates access to the CNX chaperone system substantially reduces the fraction of TMX1 co-precipitated (i.e., participating in a functional complex) with CNX. Altogether, the data in Figs. 3-4 show that CNX and TMX1 may form a functional complex, which is stabilized by client substrates. This conclusion is supported by the hampered association of TMX1 with CNX in cells with reduced protein synthesis (Figs. 4C, lane 4 and 4D, lane 3), or with defective N-glycosylation upon exposure to tunicamycin (Figs. 4C, lane 5 and 4D, lane 4).

2.5.3 Analysis of steady-state TMX1C/A:BACE501 mixed disulfides by WB

To further confirm the selective involvement of TMX1 in mixed disulfides with membrane- bound clients, BACE501 was expressed alone (Fig. 5A, lanes 1-2), with TMX1 (lanes 3-4) or with TMX1C/A (lanes 5-6). After immunoisolation of the HA-tagged bait, the immunocomplexes were separated in SDS-PAGE under non-reducing (NR, Fig. 5A, lanes 1, 4, 5) and reducing conditions (R, lanes 2, 3, 6). Proteins were then transferred to a PVDF membrane. BACE501 (Fig. 5A, lanes 1-6) or TMX1 (lanes 7-12) were revealed with HA- or TMX1-specific antibodies.

Confirming the data obtained with radiolabeled proteins (Fig. 3), the MD with the apparent MW of 90 kDa were only seen upon separation under non-reducing conditions of immunoisolates of cells co-transfected with BACE501 and TMX1C/A (Fig. 5A, lane 5 for the BACE501 component of the mixed disulfides and lane 11 for the TMX1 component). The undetectable level of immunoreactivity in cells expressing the wild type form of TMX1

(lanes 3-4 and 9-10) confirms that TMX1C/A stabilizes the otherwise short-lived reaction intermediate. Disassembly upon sample reduction revealed BACE501 and TMX1 as the constituents of the mixed disulfides (Fig. 5A, lanes 6 and 12, respectively). The absence of mixed disulfides when the same experiment was performed with the soluble BACE501 variant further supported the selectivity of TMX1 for membrane-bound substrates (Fig. 5B).

Figure 5. Characterization of TMX1C/A-BACE501 mixed disulfides by WB. A, MEFs were cotransfected with BACE501 and an empty vector (EV, lanes 1 and 2), TMX1 (3 and 4), or TMX1C/A (5 and 6). BACE501 was immunoisolated from cell lysates and immunocomplexes were analyzed under non-reducing (NR) or reducing (R) conditions. B, Same as A for BACE501Δ. red, reduced BACE; ox, oxidized BACE; DBC, disulfide-bonded complexes; MD, mixed disulfides.

2.5.4 TMX1C/A delays BACE501 maturation

Mixed disulfides are short-lived intermediates formed during the productive interaction between an oxidoreductase and its substrates 233,279. Their stabilization upon mutation of the resolving cysteine residue in the oxidoreductase’s catalytic site is expected to delay substrate release from the ER. As for all glycoproteins, release of BACE501 from the ER can be monitored by the modification of protein-bound oligosaccharides that occurs during transit in

40 the Golgi compartment, which reduces the electrophoretic mobility of the polypeptide chain and confers resistance to EndoH cleavage 290. To determine whether the co-expression of the TMX1 trapping mutant delays the attainment of the BACE501’s EndoH-resistant status,

MEFs expressing only BACE501, BACE501 and TMX1 or BACE501 and TMX1C/A were pulse labeled and chased for 10 (Fig. 6A, lanes 1, 3 and 5) or for 90 min (Fig. 6A, lanes 2, 4 and 6). After 10 min of chase, radiolabeled BACE501 expressed alone (Fig. 6B, lanes 1-2), co-expressed with TMX1 (lanes 5-6), or with TMX1C/A (lanes 9-10) is sensitive to EndoH cleavage. This is consistent with the ER localization of the newly synthesized polypeptide 281.

Figure 6. Co-expression of TMX1C/A selectively delays BACE501 maturation. A, MEFs were co-transfected with BACE501 and an empty vector (lanes 1-2), TMX1 (3-4) or TMX1C/A (5-6). Transfected cells were pulsed with 35S-methionine and -cysteine for 13 min and chased for 10 or 90 min. Ectopically expressed BACE501 was immunoisolated from cell lysates with an anti-BACE antibody. B, MEFs were co-transfected with BACE501 and an empty vector (lanes 1-4), TMX1 (5-8) or TMX1C/A (9-12). The maturation of immunoisolated radiolabeled BACE501 (i.e., the attainment of EndoH-resistant oligosaccharides showing arrival in the Golgi complex) was monitored after 10 and 90 min chase. C, Quantification of the EndoH-resistant fraction of BACE501 after 90 min chase. The error bars show standard deviations of four independent experiments. D, MEFs were co-transfected with BACE501Δ and an empty vector (EV, lanes 1-2), TMX1 (3-4) or TMX1C/A (5- 6). The panel on the left shows the disappearance of immunoisolated BACE501Δ from the cell lysates (intracellular) that correlates with its secretion in the extracellular media (right panel, Secreted). E, Quantification of secreted BACE501Δ after 90 min chase. The error bars show standard deviation of three independent experiments. Significance analyzed by paired t-test (n.s.= not significant; *= p<0.05; **= p<0.01; ***= p<0.001).

The analysis after 90 min of chase showed that when expressed alone or with the wild type form of TMX1, more than 85% of radiolabeled BACE501 displayed EndoH-resistant oligosaccharides (Figs. 6B, lanes 3-4 and 7-8, respectively and 6C). This is consistent with the efficient export to the Golgi of this protein 281. The stabilization of the TMX1:BACE501 mixed disulfides upon co-expression of TMX1C/A dramatically reduced the fraction of EndoH-resistant BACE501 to less than 40% of the radiolabeled protein (Figs. 6B, lanes 11- 12 and 6C). In contrast, and consistent with the selectivity of TMX1 for membrane-bound polypeptides (Figs. 2-4), the secretion of BACE501 (Figs. 6D, panel on the right and 6E) was unaffected by the co-expression of TMX1C/A.

2.6 Ectopic expression of the trapping mutant version of several PDI family members and BACE501 maturation

2.6.1 Selective action of TMX1C/A on BACE501

To prove the specificity of TMX1 for BACE501 maturation, we performed a pulse-chase experiment co-transfecting MEFs with BACE501 together with plasmids encoding for the trapping mutant version of the PDI family members ERdj5, ERp57, ERp72, PDI and P5 (Fig. 7A). We found that only the co-expression of the TMX1 trapping mutant substantially reduced attainment of EndoH-resistant oligosaccharide as a measure of delayed BACE501 secretion (by 40-45%, Fig. 6, Fig. 7B, lanes 3-4 and 7C). More importantly, ERdj5C/A did associate with BACE501 but only marginally delayed secretion (by 10-15%, Fig. 7B, lanes

5-6 and 7C). After 10 min of chase, the levels of radiolabeled TMX1C/A and ERdj5C/A co- precipitating with BACE501 were similar (Fig. 7B, lane 3 vs. 5), although TMX1C/A contains 35 less cysteines and methionines that incorporate S- during the pulse respect to ERdj5C/A (11 residues for TMX1C/A and 31 for ERdj5C/A). This indicates that newly synthesized TMX1C/A interacts more strongly with BACE501 than ERdj5C/A, supporting the preference of TMX1C/A for BACE501.

BACE501 co-expression with ERp57C/A (Fig. 7B, lanes 7-8 and 7C), ERp72C/A (lanes 9-10 83,203,207,250 and 7C), PDIC/A (lanes 11-12 and 7C) or P5C/A (lanes 13-14 and 7C) had no consequences. Moreover, newly synthesized ERp57C/A, ERp72C/A, PDIC/A and P5C/A did not

42 co-precipitate with BACE501. These results suggest that TMX1 plays a specific role during BACE501 folding programs.

Figure 7. PDIs trapping mutants and BACE501 maturation. A, Schematics of the PDI family members’ trapping mutants. SS, signal sequence, J-domain, Hsp70 interacting region, Trx, thioredoxin-like domain, a, active site, b, inactive site, x, linker domain. The sequence of the active sites, the position within the polypeptide sequence and the position of the active site residues are shown. B, MEFs were co-transfected with BACE501 in combination with an empty vector (EV, lanes 1-2), TMX1C/A (3-4), ERdj5C/A (5-6), ERp57C/A (7-8), ERp72C/A (9-10), PDIC/A (11-12) or P5C/A (13-14). Ectopically expressed BACE501 was immunoisolated from cell lysates with an anti-BACE antibody. The maturation of radiolabeled BACE501 was monitored as in Fig. 6. C, Quantification of the EndoH-resistant fraction of BACE501 after 90 min chase. The error bars show standard deviations of three independent experiments. Significance analyzed by paired t-test (n.s.= not significant; *= p<0.05; **= p<0.01; ***= p<0.001).

2.6.2 TMX4C/A does not affect BACE501 maturation

To further confirm the specificity of TMX1 for BACE501, we performed a pulse-chase experiment co-expressing BACE501 and the membrane-bound TMX1 paralog TMX4C/A (Figs. 8A). Phylogenetic analyses proved that TMX1 and TMX4 derive from a duplication event from a common ancestor 254. The two proteins are highly similar in terms of topology, active site sequence and ER localization signal (see Introduction, section 1.5.2.4.2). Moreover, in vitro assays confirmed that both enzymes possess reductase activity 256,262.

Figure 8. TMX4 trapping mutant and BACE501 maturation. A, Schematics of TMX4 trapping mutant. SS, signal sequence, Trx, thioredoxin-like domain, TM, transmembrane region. The sequence of the active site and the position of the active site residues are shown. B, MEFs were co-transfected with BACE501 in combination with an empty vector (EV, lanes 1-2), V5- tagged TMX1C/A (lanes 3-4) or TMX4C/A (lanes 5-6). Ectopically expressed BACE501 was immunoisolated from cell lysates with an anti-BACE antibody. C, Quantification of the EndoH-resistant fraction of BACE501 after 90 min chase. D, Same as B for immunoisolation from cell lysates with an anti-V5 antibody.

As in the case of ERdj5C/A co-expression, TMX4C/A did associate with BACE501 but only marginally delayed its secretion (by 10%, Fig. 8B, lane 6 and 8C). Newly synthesized

TMX1C/A and TMX4C/A contain the same number of residues that are radiolabeled during the

44 pulse (11 for TMX1C/A and 10 for TMX4C/A). Immunoisolation of TMX1C/A and TMX4C/A form MEFs lysates with an anti-V5 antibody after 10 min of chase revealed that cells contain more TMX4C/A than TMX1C/A (Fig. 8D, lanes 3 and 5). Importantly, the level of the ER form of BACE501 co-precipitating with TMX1C/A was much higher than that co-precipitating with

TMX4C/A, suggesting that TMX4C/A, contrarily to TMX1C/A, weakly interacts with BACE501

(Fig. 8D, lane 3 vs. 5). This result supports the specificity of TMX1C/A for BACE501. Moreover, consistent with what has already been described in Figs 6-7, after 90 min of chase only the co-expression of TMX1C/A caused a prominent reduction of BACE501 secretion (Fig. 8B, lane 4 vs. lane 6 and 8C). From this experiment we conclude that the specificity of TMX1 for BACE501 is not solely given by their common localization at the ER membrane.

2.6.3 Selective action of TMX1C/A vs. non-selective effect of ERdj5C/A

According to our data, TMX1C/A shows preference for membrane-bound polypeptides (Figs.

2-4). In line with that, TMX1C/A does not interact via MD with the soluble BACE501 and does not affect BACE501 secretion (Figs. 6D-E). A mass spectrometry analysis performed using ERdj5C/A to identify its endogenous substrates, revealed that ERdj5C/A did interact with both soluble and membrane-bound substrates, without operating a topology-based 250 discrimination . Interestingly, here we found that ERdj5C/A, as in the case of BACE501, also associated with the soluble BACE501Δ, thereby reducing (by 20%) the secretion of this soluble model protein (Figs. 9A, lanes 5-6 and 11-12, 9B). This result is in agreement with previous indications and shows that in contrast to TMX1, ERdj5 does not apparently distinguish between the membrane-bound and the luminal version of the same maturing substrate. Moreover, it appears that TMX1C/A potently affects BACE501 maturation (Figs. 6-

7-8) while ERdj5C/A only partially intervenes during BACE501 and BACE501 folding programs (Figs. 7 and 9).

Figure 9. Trapping mutants of several PDI family proteins and BACE501Δ maturation. A, MEFs were co-transfected with BACE501Δ in combination with an empty vector (EV, lanes 1-2), TMX1C/A (lanes 3- 4), ERdj5C/A (lanes 5-6), ERp57C/A (lanes 7-8), ERp72C/A (lanes 9-10), PDIC/A (lanes 11-12) or P5C/A (lanes 13-14). Ectopically expressed BACE501Δ was immunoisolated from cell lysates with an anti-BACE antibody. The upper panel shows the disappearance of BACE501Δ from the cell lysates (intracellular) that correlates with its secretion in the extracellular media (lower panel, Secreted). B, Quantification of secreted BACE501Δ after 90 min chase. The error bars show standard deviations of two independent experiments.

2.7 Role of TMX1C/A in ERAD

2.7.1 TMX1C/A preferentially associates with membrane-bound folding-defective polypeptides

Several studies demonstrated that PDI family proteins intervene to unfold ERAD substrates facilitating their linearization and consequently their retro-translocation across the ER membrane and their degradation by cytosolic proteasomes 130,133,291,292 (see Introduction, section 1.2.3). BACE457 and BACE457 are well characterized glycosylated ERAD substrates that contain three disulfide bonds 130.

To investigate whether TMX1 intervenes in ERAD, we first analyzed the association of the TMX1 trapping mutant with a set of membrane-bound and soluble ERAD substrates (Fig. 10A). BACE457 and BACE457, already examined before (Figs. 2B-C, lanes 5-6) were included as positive and negative controls for TMX1 interaction, respectively. MEFs were transfected with plasmids for the expression of BACE457 (Fig. 10B, lane 1), BACE457 (lane 2), the non-glycosylated versions of BACE457 (BACE457N, lane 3) or BACE457∆ (BACE457N, lane 4), the folding-defective transport-incompetent soluble α-1- 293 antitrypsin variant NHK (lane 5) or the chimeric membrane-bound version NHKCD3δD6A 105 (lane 6). Notably, NHK does not possess disulfide bonds as its ectodomain contains only one cysteine. This residue promotes the formation of covalent homodimers, causing NHK retention within the ER lumen. Reduction of these covalently-bound homodimers precedes NHK degradation 294 and requires the intervention of a reductase.

MEFs were detergent-solubilized and the ectopically expressed HA-tagged BACE and NHK variants were immunoisolated from post-nuclear supernatants. The immunocomplexes were separated in SDS-PAGE and transferred to PVDF membranes. The presence of BACE and NHK variants was revealed by WB with anti-HA antibodies (Fig. 10B, upper panel). The association of TMX1C/A was assessed, in the same PVDF membrane, upon decoration of the membrane with anti-TMX1 antibodies (Fig. 10B, lower panel). In agreement with our previous results, we found that TMX1C/A associates with the membrane-bound versions of BACE (i.e., BACE457 and non-glycosylated BACE457N, Fig. 10B, lower panel, lanes 1 and

3). TMX1C/A association is substantially reduced for the soluble variants BACE457∆ and BACE457N∆ (lanes 2 and 4, respectively). Moreover, in line with a preferential association of TMX1 with membrane-bound substrates, TMX1C/A strongly associated with NHK only when tethered to the ER membrane via a membrane-anchor (Fig. 10B, lane 6 vs. lane 5).

Figure 10. TMX1C/A preferentially associates with membrane-bound folding-defective substrates A, Schematics of the model polypeptides used in panels B. B, MEFs co-transfected with V5-tagged TMX1C/A in combination with HA-tagged BACE457 (lane1), BACE457Δ (lanes 2), BACE457N (lane 3), BACE457N (lane 4), NHK (lane 5) or NHKCD3δD6A (lane 6). The HA-tagged model substrates were immunoisolated from cell lysates. The upper panel shows a WB with an anti-HA antibody to reveal the model substrates, the lower panel a WB with a TMX1-specific antibody to reveal TMX1C/A.

These results suggest that TMX1 does not discriminate between glycosylated and non- glycosylated misfolded polypeptides. Rather, as in the case of folding-competent polypeptides, it preferentially recognizes membrane-bound proteins.

2.7.2 TMX1C/A intervenes during BACE457 disposal

2.7.2.1 TMX1C/A forms mixed disulfides with BACE457

To confirm that the association between TMX1 and the folding-defective membrane-bound BACE457 was indeed a covalent association mediated by mixed disulfides, MEFs were co- transfected with plasmids encoding for BACE457 and TMX1C/A (Figs. 11A-B, lanes 1-2). After immunoisolation of BACE457, the immunocomplexes were separated in SDS-PAGE under both non-reducing (NR, lanes 1) and reducing conditions (R, lanes 2). Proteins were then transferred to a PVDF membrane. BACE457 (Fig. 11A, lanes 1-2) or TMX1 (Fig. 11B, lanes 1-2) were revealed with BACE- or TMX1-specific antibodies, respectively. High MW DBC and a MD with the apparent MW of 90 kDa were seen upon separation under non- reducing conditions (Fig. 11A, lane 1 for the BACE457 component and Fig. 11B, lane 1 for the TMX1 component). High MW BACE457-positive complexes were also present upon sample reduction (Fig. 11A, lane 2): importantly, these complexes did not contain TMX1C/A (Fig. 11B, lane 2), representing BACE457-containing protein aggregates. A part from this

DTT-resistant fraction of BACE457, sample reduction allowed dissociation of DBC and MD complexes containing BACE457 and TMX1C/A as shown by the appearance of intense bands of about 50 kDa (BACE457) and 35 kDa (TMX1C/A, Figs. 11A-B, lanes 2). Thus, as with the folding-competent BACE501, TMX1C/A is able to establish mixed disulfides with the membrane-bound folding-defective splice variant BACE457.

Figure 11. Characterization of TMX1C/A:BACE457 mixed disulfides by WB. A, MEFs were co-transfected with BACE457 and TMX1C/A. BACE457 was immunoisolated from cell lysates and immunocomplexes were analyzed under non-reducing (NR, lanes 1) or reducing (R, lanes 2) conditions. BACE457 was revealed with a BACE-specific antibody. B, Same as A for TMX1C/A revealed with a TMX1- specific antibody. DBC, disulfide-bonded complexes; MD, mixed disulfides.

2.7.2.2 TMX1C/A forms mixed disulfides with newly synthesized BACE457

To better characterize the interaction between TMX1 and BACE457, MEFs expressing

BACE457 or co-expressing BACE457 and TMX1C/A were pulse labeled and chased for 10 min (Fig. 12A, lanes 1 and 3). BACE457 was immunoisolated from detergent extracts and complexes were analyzed in non-reducing (Fig. 12A, NR, left panel) and reducing SDS- PAGE (Fig. 12A, R, right panel). In the non-reducing gel, BACE457 expressed alone (Fig.

12A, left panel, lanes 1) or in combination with TMX1C/A (lanes 3) was separated in a series of radiolabeled bands corresponding to various oxidation forms (partially oxidized, pOx; fully oxidized, fOx;). This is confirmed by the fact that upon reduction, all these forms collapsed into a single band, corresponding to the reduced form of BACE457 (Fig. 12A, right panel). Notably, in MEFs chased for 10 min, where the membrane-bound BACE457 was co-transfected with TMX1C/A, separation of BACE immunoisolates under NR conditions showed the presence of an intense radiolabeled band with an apparent MW of about 90 kDa

(MD, Fig. 12A, left panel, lane 3) and of DBC of more than 200 kDa (Fig. 12A, left panel, lane 3).

Figure 12. TMX1C/A establishes mixed disulfides with newly synthesized BACE457. A, MEF were co-transfected with EV or 35 TMX1C/A (lanes 1-4). S- methionine and -cysteine radiolabeled model substrates were immunoisolated from cell lysates with anti-BACE antibodies. The immunocomplexes were separated under non-reducing (NR) and reducing (R) conditions. Transfected cells were chased for 10 or 150 min. B, Quantification of the ER-resident fraction of BACE457 after 150 min chase. C, Same as A for cells transfected with BACE457. D, Same as B for BACE457. pOx, partially oxidized BACE; fOx, fully oxidized BACE; Red, reduced BACE; DBC, disulfide-bonded complexes; MD, mixed disulfides.

The molecular weight of the 90 kDa band corresponded to a 1:1 BACE457 (MW of about 50 kDa) and TMX1C/A (MW of about 35 kDa) complex. Indeed, as shown in Figure 11, sample reduction dissociated the radiolabeled 90 kDa polypeptide in the two components (Fig. 12A, right panel, lane 3). Remarkably, upon MD dissociation, a band of about 97 kDa corresponding to CNX was revealed. We think that similarly to what we saw for the folding-

50 competent BACE501, CNX might work with TMX1C/A in the disposal of the folding- defective BACE457. In agreement with data shown above, the soluble BACE457 did not associate with

TMX1C/A (Fig. 12C, left panel, lane 3).

2.7.2.3 TMX1C/A delays BACE457 degradation

The stabilization of the complex between TMX1 and BACE457 achieved by the trapping mutant TMX1C/A is presumed to slow down BACE457 degradation and promote its ER retention. To assess whether the co-expression of TMX1C/A delays the degradation of

BACE457, MEFs expressing BACE457 or co-expressing BACE457 and TMX1C/A were pulse labeled and chased for 10 (Fig. 12A, lanes 1 and 3) or for 150 min (Fig. 12A, lanes 2 and 4). The analysis after 150 min of chase showed that when BACE457 was expressed alone, about 20% of radiolabeled BACE457 was still retained intracellularly (Figs. 12A, right panel, lane 2 and 12B). This is consistent with an efficient degradation of the protein after 150 min of chase. The stabilization of the TMX1:BACE457 MD upon co-expression of TMX1C/A determined an increase of the residual, radiolabeled BACE457 (up to 50%, Figs. 12A, right panel, lane 4 and 12B). Again, TMX1:BACE457∆ MD were not formed and the degradation of BACE457 was unaffected by the co-expression of TMX1C/A (Figs. 12C-D). These results confirm that TMX1 is specifically involved in the degradation of the membrane-bound folding-defective BACE457.

3. Discussion

The mammalian ER contains 23 PDI family members (Table 1) 62 that are responsible for the catalysis of thiol-disulfide exchange reactions during oxidative protein folding and during preparation for degradation of misfolded polypeptides.

Since PDI itself has been reported to catalyze all the three redox reactions (oxidation, reduction and isomerization) both in vitro and in vivo 295,296, understanding why so many PDIs are present in the ER is a question that deserves particular attention. Above all, the study of the topological diversification (soluble vs. membrane-bound PDIs) shown by PDI members might be of particular interest. The membrane-tethered vs. soluble localization of individual PDIs can indeed dictate their in vivo specificity and substrate selection. For the two PDIs included into the OST complex, MagT1 and Tusc3, the membrane anchors confer the best topology to assist the efficient glycosylation of newly synthesized polypeptides entering the ER by retarding formation of intramolecular disulfides 52,251. Likewise, the functional specificity of the membrane-bound PDIs belonging to the TMX subgroup might be addressed to membrane-proximal folding and ERAD processes. The preferential redox activity for three of the five TMX members has been investigated using different in vitro assays (see section 3.2.2) whereas the characterization of their in vivo function remains poorly described 206. Thus, the characterization of this peculiar subgroup still represents an important goal in order to address PDIs functions.

During my PhD, I focused my attention on TMX1. I found that this ER-resident PDI is not up-regulated during ER stress, it forms a functional complex with the ER lectin CNX and that it preferentially interacts with membrane-bound substrates regardless of their glycosylation status or folding competence. The trapping mutant TMX1C/A retains in the ER membrane-bound polypeptides thereby retarding secretion of folding-competent and degradation of folding-defective ones. All in all, the data presented in the thesis identify TMX1 as the first example of a topology-specific client protein redox catalyst in living cells.

3.1 PDI proteins: an overview

PDI proteins display different domain architecture, active-site compositions, enzymatic properties, interacting partners, sub-organellar localization and tissue distribution 83,206,207. The domain architecture changes between PDI members, with many proteins possessing only the active a domains, only the inactive b domains or both. The number and position of these domains, together with the presence of additional functional ones, such as Hsp-binding (J- domain) or transmembrane (t-domain) domains, obviously contribute to the important structural and functional diversity among PDI family members. Moreover, the very same domain may change in terms of sequence, thus assuming a completely different functional connotation. An exemplification of this aspect is the difference that exists between the b’ domains of PDI and ERp57, the former binding substrates, the latter associating with the ER lectins CNX and CRT (see section 3.3). Another example is given by the different amino acidic composition of the CXXC active site motifs contained in the a domains. By looking at the amino acids within the CXXC motifs (Table 1), it is possible to predict the preferential redox activity of each PDI member, although the physiological conditions in which they work in might substantially affect the enzyme’s redox function. Nevertheless, select PDI proteins work adequately when located in their proper sub-compartmental domains or included in specific functional complexes. The CNX/CRT-determined specificity of ERp57 in the context of glycopolypeptides’ folding is probably the most prominent case (see section 3.3).

Considering this extremely intricate scenario, in vitro and in vivo studies represent essential systems for PDIs’ functional dissection (see sections 3.2.1 and 3.3). Actually, although such a great number of PDI proteins suggests extensive functional redundancy, accumulating evidences prove that individual PDIs display exquisite substrate specificity207,212,244.

3.2 In vitro characterization of PDI proteins

3.2.1 Assays to detect oxidoreductase’s activity

Commonly, the functional characterization of PDIs starts from the in vitro assessment of the redox potential and of the thiol-disulfide exchange reactions catalyzed.

In principle, all active site-containing PDIs might catalyze substrate oxidation, reduction and isomerization, however, the relative catalytic efficiency differs among individual PDIs. The redox potential measures the relative stability of the oxidized (disulfide) vs. reduced (di-thiol) state of the PDIs’ active site. Many intrinsic factors are involved in the modulation of the redox potential, such as the pKa of the N-terminal cysteine, the two inner XX residues of the active site and structural constraints 205. Proteins containing CGHC active sites, such as PDI, ERp57, ERp72, P5 and ERp46, are thought to work prominently, but not exclusively, as oxidases. Instead, PDIs containing proline residues between the active site cysteines, such as ERdj5, TMX1 and TMX4, might preferentially work as reductases. The in vitro assessment of the redox potential for each PDI enzyme helps in the prediction of the individual function, however, environmental conditions in the ER lumen such as redox environment and pH affect the redox potential value. Recently, Hudson and colleagues reported a list of the PDI proteins whose redox potential has been estimated. Importantly, they also included the predicted redox state assuming the ER redox poise at -225 mV 297.

In vitro assays to understand PDI activity represent another important tool to study their functional diversification. In order to do that, three main classes of assays are commonly employed, according to the redox reaction of interest 205,298. The first class is based on the gain of activity of a model substrate, such as RNase A, and is employed to study the oxidative refolding of the reduced form of the model substrate upon addition of the select PDI member under investigation. For an enzyme that possesses oxidase activity, RNase refolding will be observed. The second class of assays involves changes in the biophysical properties of a model substrate. The insulin reduction assay belongs to this class and is commonly used to study PDIs’ reductase activity. Reduction of insulin results in protein aggregation and involves changes in light adsorption. Finally, the third class relies on the recovery of the functional activity of a model substrate. As an example, an inactive form of

RNase A containing scrambled disulfides is used to measure the isomerase activity of PDI proteins, where isomerization will result in RNase A conversion to its active state 298.

An important limitation of these assays is that they are performed using specific portions of the proteins of interest, i.e. the thioredoxin-like luminal domains, rather than full-length proteins. Moreover, these experiments are performed in test tubes, without considering the natural PDIs’ sub-cellular compartmentalization and molecular environment, using PDIs and model substrates in non-physiological concentrations, employing model substrates that are not natural physiological PDI substrates. Thus, the resulting picture might be a distortion of the enzymes’ physiological functions. Coupling in vitro data with in vivo analysis represents the unavoidable step to achieve a more solid result.

3.2.2 In vitro characterization of TMXs

The TMX subfamily includes three members that are catalytically active (TMX1, TMX3, TMX4), one that is supposed to be a natural trapping mutant, with a N-terminal active site’s cysteine able to perform the nucleophilic attack but unable to resolve the resulting mixed disulfide due to the absence of the C-terminal active site’s cysteine (TMX5) and an inactive member (TMX2).

While for TMX1 there are no evidences about its redox potential, by expressing the thioredoxin-like domains of TMX3 and TMX4, their redox potential has been measured as - 157 and -170 mV, respectively 261,262.

In order to assess the redox activity of these enzymes, the luminal thioredoxin-containing portions of TMX1, TMX3 and TMX4 have been used to perform the aforementioned in vitro assays (see section 3.2.1). The TMX1 27-180 peptide has been found to catalyze the isomerization of misplaced RNase A disulfides, leading to RNase reactivation 256, while the TMX4 35-185 peptide did not show any isomerase activity 262. This result constitutes the first indication that the two paralogues do not have equivalent functions.

Moreover, TMX1 27-180 peptide was found to be able to reduce insulin and promote an increase in turbidity, proving the in vitro reductase activity for TMX1 252. Similarly, TMX4

35-185 peptide was also proved to reduce insulin, confirming the in vitro reductase activity for TMX4 262. Taken together, these in vitro analyses show that TMX1 possesses reductase and isomerase activities, while TMX4 is in principle only able to catalyze disulfide reduction. Importantly, the proven reductase activity for both enzymes agrees with the prediction done by simply looking at the active site composition of TMX1 and TMX4 (CPAC and CPSC, respectively).

A fluorescent-based in vitro assay performed with the luminal portion of TMX3 (25-373 residues) proved that TMX3 catalyzes the oxidation of a model peptide, although at a lesser extent compared to PDI. This result also agrees with the active site amino acidic composition predicted to confer to TMX3 the in vitro oxidase activity 261.

Importantly, these data point out that as in the case of luminal PDIs, also TMX proteins possess distinct oxidoreductase activities.

3.3 In vivo studies: a question of redundancy and specificity

The dissection of the physiological role of each PDI family member implies its analysis in living cells. Knock-out or knock-down of individual PDIs represent a common strategy to study which defects derive from specific protein’s depletion. As in the case of PDI, constitutive knock-out might results in cell death 230, so knock-down by siRNA is commonly used to investigate the role of PDI enzymes in proteome maintenance 207. The limitation of this approach derives from the residual protein amount after gene silencing, which can be sufficient to catalyze the target reactions, preventing the detection of clear phenotypes 207 and, as mentioned above, the redundancy of the system.

PDI depletion is well-tolerated by mammalian cultured cells and only causes slight defects in the oxidative folding of newly synthesized proteins such as albumin, α-fetoprotein, 207 transferrin and α2-HS-glycoprotein . Beyond the fact that PDI is considered the most prominent ER oxidoreductase, this result is surprising taking into account that PDI is thought to be largely responsible for the ER transfer of oxidizing equivalents via Ero1 299. The minor phenotype associated with PDI depletion confirms that alternative routes for both oxidative

56 protein folding and oxidizing equivalents’ transfer do exist in the mammalian ER and that a certain degree of redundancy between PDI proteins warrants maintenance of proteostasis. Accordingly, many other examples of hardly detectable phenotypes in cell lines derived from knockout mice for select PDI proteins have been reported 134,207,212,231,300.

It was initially thought that ERp57 might be responsible for the compensation of PDI depletion. Although ERp57 and PDI do show a certain degree of overlap in terms of substrate specificity 207, PDI depletion affects oxidative folding of both glycosylated and non- glycosylated proteins, whereas ERp57 depletion seems to only affect maturation of select glycoproteins 207,212. PDI’s specificity toward substrates seems to be structural-dependent and is principally given by its b’ domain, which directly interacts with hydrophobic patches displayed by the substrates’ chains, irrespective of their glycosylation status 214. Moreover, since PDI forms functional complexes with the ER chaperones BiP and GRP94, a chaperone- driven substrate recognition might also occur 85. Also in the case of ERp57 substrate specificity is determined by its b’ domain, which greatly differs from that of PDI. The b’ domain of ERp57 does not recognize hydrophobic regions. Rather it interacts non-covalently with CNX and CRT 86, thus conferring to ERp57 the specificity toward glycopolypeptides. Among glycopolypeptides, ERp57 prefers proteins possessing little secondary structure and domains reach in disulfides, suggesting a structural-dependent specificity 244.

Overall, PDI and ERp57 are the best-studied ER oxidoreductases. They possess distinct substrate preference but also intervene at different stages during the polypeptides folding process 207. This scenario seems plausible if considering the results obtained in ERp57 deficient mammalian cells 212. It has been shown indeed that ERp57 depletion does not affect co-translational folding of newly synthesized polypeptides but is required during posttranslational isomerization of mispaired disulfides. Moreover, the severity of the phenotype observed paralleled the degree of dependency of the substrates on CNX/CRT. Interestingly, among the clients identified for ERp57, some have been found to strictly depend on ERp57, while others were more promiscuous, being equally well assisted by ERp72 when ERp57 was depleted 212. Additional studies revealed that ERp57 depletion in B cells did not impair general oxidative protein folding and did not alter the rearrangement of disulfides in the ERp57 substrate MHC class I, rather ERp57 lack had consequences on the MHC class I-

57 peptide loading complex assembly and stability 301. These data further reinforce the redundant aspect of the PDIs’ system in the context of oxidative folding.

As in the case of ERp57 that specifically works on CNX/CRT substrates, ERdj5 and P5 are thought to possess chaperone-mediated specificity toward substrates 83,133. According to the literature, the role for ERdj5 in both folding and ERAD is decided by its functional association with ER resident factors. Specifically, ERdj5 reductase/isomerase activity is addressed to EDEM1- and BiP-clients 133. On the same line, P5 also interacts with BiP, thus leading to hypothesize that it works on BiP-clients 83.

3.4 Functional characterization of TMX1

The functional characterization of TMX1 has been initiated in 2001 by the group of Yodoi and colleagues. A part from the in vitro evidences showing that it possesses both reductase and isomerase activities (see section 3.2.2), they showed that TMX1 forms mixed disulfides with MHC-I heavy chain, as already shown for PDI and ERp57 257. They also reported that TMX1 was principally in the reduced form in vivo 257 and that TMX1 knock-out mice only exhibited susceptibility to liver inflammation upon lipopolysaccharide and D-galactosamine treatments 258.

MEFs derived from these knock-out mice were used in our laboratory to analyze the role of TMX1 during protein folding and ERAD. As already shown by Yodoi and colleagues 258, we were not able to detect any phenotype looking at maturation or degradation of model polypeptides (data not shown). We reasoned that adaptation to TMX1 loss could occur in cell lines. To identify endogenous substrates of TMX1, we therefore decided to make use of a trapping mutant version of TMX1. A similar approach has been used to trap in vivo endogenous substrates of other PDI family members 83,244. According to the reductase/isomerase activity proposed by in vitro studies, we decided to use the C-terminal active site trapping mutant that allows the stabilization of substrates on the reductive pathway 211. This approach allowed us to establish the specificity of TMX1 for membrane-bound cysteine-containing polypeptides.

Protein topology has been reported to constitute a key factor in determining engagement of folding, quality control, and degradation pathways. One example is represented by the lectin chaperone system, in which the soluble CRT and the membrane-bound CNX show different specificity for newly synthesized polypeptides that enter the ER 302-306. Beyond the slight difference they exhibit toward glycans according to their position within the polypeptide chains, the distinct topology of the two lectins confers the preference for different substrates. Indeed, the attachment of CRT to the ER membrane or the detachment of CNX resulted in the reversion of their substrate specialization 304,306.

TMX1 forms functional complexes with CNX and these complexes are stabilized in presence of endogenous transmembrane substrates undergoing maturation or in presence of the ectopic folding-competent transmembrane BACE501. As in the case of ERp57 that specifically works on CNX/CRT substrates, we propose that TMX1 possesses specificity toward CNX substrates that are bound to the ER membrane. Indeed, inhibition of endogenous or ectopic transmembrane protein association with CNX leads to disassembly/destabilization of functional complexes between CNX and TMX1, as in the case of functional complexes between CNX and ERp57 86,244 or between CNX and the peptidyl-prolyl isomerase CypB 84. Importantly, TMX1 does not associate with CRT 257 and does not bind to the soluble version of BACE501. Taken together, these data agree upon the existence of a topology-determined functional complex located at the level of the ER membrane that seems to be required for proper oxidative folding of membrane-anchored polypeptides.

In addition, topology-based preferences have also been shown for supramolecular complexes that mediate disposal of misfolded polypeptides, where soluble misfolded products display strict dependency for members of the HRD1-based dislocation complex 122,148. TMX1’s implication in ERAD has been hypothesized due to its involvement in ricin’s reduction 167. Importantly, ricin does associate with CNX but not with CRT 167. On this line, we found that TMX1 is involved in the disposal of the folding-defective membrane-bound BACE457. Importantly, we identified a complex containing TMX1, BACE457 and CNX, leading to extend the role of functional TMX1:CNX complexes in ERAD as well.

Thus, combining previous evidences with our data, it is reasonable to propose that functional complexes located both in the ER lumen and at the ER membrane do exist to regulate

59 maturation, quality control and disposal of newly synthesized polypeptides entering the ER lumen.

3.5 Functional evidences about the other TMX proteins

In 2005, Ellgaard and colleagues performed a pioneering study on TMX3. They reported that TMX3 was ubiquitously expressed in various human tissues and that it was not regulated by ER stress 261. In order to identify putative endogenous substrates for the enzyme, they made use of the C-terminal active site trapping mutant of TMX3. Importantly, in agreement with its proposed oxidase activity (see section 3.2.2), the employment of this construct was unable to reveal substrates for the enzyme 261. An aim for future works will be the assessment of TMX3 substrates by using the proline trapping mutant version of TMX3, thus stabilizing in principle substrates on the oxidative pathway.

A functional analysis of TMX4 performed by Nagata and colleagues revealed that TMX4 interacts with CNX and ERp57, thus a role for in oxidative glycoprotein folding was hypothesized 262. In the same study, according to its in vitro reductase activity (see section 3.2.2), a possible role as ERAD reductase was also investigated by looking at NHK degradation’s rate in TMX4 knockdown cells. In these experimental conditions, NHK degradation was unaffected, suggesting that TMX4 might not be required for NHK dimers reduction or that other PDIs were able to compensate the loss of TMX4 262. Future experiments using the trapping mutant version for TMX4 would help in the assessment of its substrate specificity. To us, it would be particularly interesting the comparison between the paralogues TMX1 and TMX4, trying to dissect the reason for their duplication.

TMX2 and TMX5 are the less-characterized TMX members. To date, there are no evidences regarding their in vitro or in vivo functions. TMX2 displays an atypical topology and its active site SNDC, putatively located in the cytosol, is presumed not to have oxidoreductase activity. On the contrary, TMX5 exhibits the CRFS active site as the unconventional ERp44 does. The presence of a single cysteine in the active site of ERp44 has been proposed to confer to the enzyme an important role during thiol-mediated retention 224. An interesting

60 parallel between the soluble ERp44 and the membrane-bound TMX5 could represent indeed an interesting topic to investigate.

All in all, we think that the assessment of the functional role of the other four TMX proteins represents the physiologic continuation of this work. As discussed throughout this thesis, the employment of the appropriate trapping mutant version and the comparison of the substrate subsets of each TMX member would allow the functional dissection of the TMX family in terms of activity and specificity. Moreover, the characterization of functional complexes for each TMX protein would help the description of the TMXs’ way of working.

4. Materials and methods

Antibodies, expression plasmids, and inhibitors

Antibodies to TMX1 and HA were from Sigma-Aldrich (Buchs, Switzerland) and antibody to V5 from Invitrogen (Lucerne, Switzerland). The rabbit polyclonal antisera 855 and 809 (used to recognize membrane-bound and soluble BACE variants, respectively) were kind gifts of P. Paganetti (Neurocentro Svizzera italiana, Taverne, Switzerland). The rabbit polyclonal antibody to CNX was kind gift of. A. Helenius (ETH, Zurich). Genes encoding the HA-tagged TMX1 and TMX1C/A were subcloned in pCDNA3. The β1-tagged TMX1 and

TMX1C/A variants were created by replacing the HA tag with an EFRH epitope, which is recognized by a monoclonal β1 antibody (Paganetti et al., 2005). Plasmids encoding the membrane-bound and soluble BACE are described in Molinari et al. 2002. Plasmids encoding for A1AT and NHK soluble and transmembrane variants are described in Merulla et al. 2015. V5-tagged ERdj5C/A-, ERp57C/A-, ERp72C/A-, PDIC/A-, and P5C/A-expressing vectors are described in Jessop et al. 2009 and Oka et al. 2013.V5-tagged TMX4C/A- expressing vector was synthesized by GenScript. Tunicamycin, cycloheximide, and CST (Sigma-Aldrich) were used at final concentrations of 5 μg/ml, 50 μg/ml, and 1 mM, respectively.

Cell lines and transient transfection

MEFs were cultured in DMEM supplemented with 10% fetal bovine serum. Cells grown on 3.5/6 cm culture dishes were transfected with 3 μg/6 μg of total plasmid DNA, using the jetPrime reagent (Polyplus transfection). Experiments were performed 17 h after transfection.

Cell lysis and western blots

Cells were washed with phosphate-buffered saline (PBS) containing 20 mM N- ethylmaleimide (NEM) for 1 min and then lysed with 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS; Anatrace) in 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES)–buffered saline, pH 6.8, supplemented with 20 mM NEM and protease inhibitors for 20 min on ice. Postnuclear supernatants (PNSs) were collected by centrifugation at 10,000 × g for 10 min. Samples were denatured and reduced in dithiothreitol (DTT)-containing sample buffer for 10 min at 65°C and separated by SDS– PAGE. Proteins were transferred to PVDF membranes with the Trans-Blot Turbo Transfer System (Bio-Rad, Cressier, Switzerland). Membranes were blocked with 10% (wt/vol) nonfat dry milk (Bio-Rad) and stained with the aforementioned primary antibodies and horseradish peroxidase–conjugated secondary antibodies. Membranes were developed using the Luminata Forte ECL detection system (Millipore, Schaffhausen, Switzerland), signals were detected with the ImageQuant LAS 4000 system in the standard acquisition mode (GE Healthcare Life Sciences, Glattbrugg, Switzerland), and bands were quantified using the Multi Gauge Analysis tool (Fujifilm). For each antigen, the linearity of the detected signal range was ensured with appropriate loading controls.

Metabolic labeling, immunoprecipitations, and EndoH treatment

Cells were pulse labeled with 0.1 mCi of [35S]-methionine/cysteine and chased in DMEM supplemented with 5 mM unlabeled methionine and cysteine. Cells were lysed as described, and PNS and extracellular medium were collected by centrifugation at 10,000 × g for 10 min and precleared with protein A beads (Sigma-Aldrich; 1:10 [wt/vol] swollen in PBS) for 1 h at 4°C. Immunoprecipitation was performed with protein A beads and specific antibody overnight at 4°C. After extensive washing of the immunoprecipitates with 0.5% CHAPS, beads were resuspended in sample buffer without (nonreducing conditions) or with DTT (reducing conditions) and denatured for 10 min at 65°C. Samples were subjected to SDS– PAGE. After exposure of the gels to autoradiography films (GE Healthcare, Fuji), films were scanned with the Typhoon FLA 9500 software, version 1.0. Bands were quantified using ImageQuant software (Molecular Dynamics, GE Healthcare). For EndoH (New England

Biolabs, Allschwil, Switzerland) treatment, immunoisolated proteins were split into two aliquots and incubated in the presence or absence of 5 mU of EndoH for 2h at 37°C. Samples were then analyzed by reducing SDS–PAGE.

Indirect immunofluorescence

MEFs transfected with plasmids for the ectopic expression of TMX1 and TMX1C/A were plated on glass coverslips prepared for immuno-fluorescence analysis with standard protocols used in our laboratory. Nuclear staining with DAPI and ER-staining with anti-CNX antibody were performed. Microscopy images were collected using a laser scanning confocal microscope (Leica DI6000 microscope stand, SP5 scan head) equipped with a HCX PL APO CS 100x1.44 oil UV objective. ImageJ software (version 1.45r) was used for image analysis and processing.

RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)

The isolation of total RNA from cells was performed with the GenElute Mammalian Total RNA Miniprep Kit (SIGMA) according to the manufacturer’s instructions. One μg of RNA was used for cDNA synthesis with oligo(dT) and the SuperScript II reverse transcriptase (Invitrogen) according to the instructions of the manufacturer.

Quantitative real-time PCR (qRT-PCR)

For each reaction, 10µl of Power SYBR Green PCR Master Mix (Applied Biosystems) and 4 µl of milliQ sterile water were added to 5 µl cDNA together with 1 µl of 10µM forward and reverse primer mix for the transcript of interest. A master mix of SYBR Green and water (mix1) was prepared accordingly to the number of samples and added to the adequate amount of cDNA (mix2). 1 µl of the primer mix for the genes of interest was pipetted in each well of a 96-Well reaction plate (MicroAmp Fast Optical 96-Well Reaction Plate with Barcode (0.1mL), Applied Biosystems)) and spun down. 19 µl of mix2 were added to each corresponding well and briefly spun down. The plate was sealed, vortexed and centrifuged.

Samples were loaded as duplicates. Quantitative Real-Time PCR was performed using a 7900HT Fast Real-Time PCR System. The housekeeping gene Actin was used as reference. Data were analyzed using the SDS 2.2.2 software.

Mass spectrometry

Confluent MEFs transfected with an empty vector or transfected with HA-tagged TMX1C/A were rinsed with PBS and 20 mM NEM. Cells were lysed with 2% CHAPS (Anatrace) in HEPES-buffered saline, pH 6.8, supplemented with 20 mM NEM and protease inhibitors for 20 min on ice. Immunoisolates were washed three times with lysis buffer. Mass spectrometry analysis was performed at the Protein Analysis Facility, University of Lausanne, Lausanne, Switzerland.

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6. Appendix

Curriculum vitae

PERSONAL DATA

Name: Brambilla Pisoni First name: Giorgia Cell phone: +41 798278075 Email address: [email protected] Skype contact: giorgiabp86 Nationality: Italian Birthday & birthplace: February 8, 1986 - Milan (Italy)

EDUCATION

• January 2013 – April 2016: PhD student at the International PhD School in Microbiology and Immunology, ETH Zürich (Switzerland). Experimental work at the Institute for Research in Biomedicine (IRB), Bellinzona (Switzerland).

Thesis title: “In vivo characterization of TMX1: TMX1 preferentially acts upon transmembrane polypeptides”.

Discussion date: March 10, 2016.

Advisors: Maurizio Molinari, PhD; Markus Aebi, PhD; Roberto Sitia, MD, PhD; Paola Picotti, PhD.

• September 2012 – December 2012: Internship. Protein Folding and Quality Control lab directed by Dr. Maurizio Molinari. Institute for Research in Biomedicine (IRB), Bellinzona (Switzerland).

• March 2010- March 2012: M.Sc. in Molecular Biology. University of Milan, Milan (Italy). Experimental work at University of Milan, section of Plant Physiology, Milan (Italy).

Thesis title: “ACA12, a Ca++ATPase IIB of A. thaliana: intracellular localization and evidences of heterologous expression”.

Final Mark: 109/110; discussion date: March 8, 2012.

Advisors: Maria Ida De Michelis, PhD; Maria Cristina Bonza, PhD.

• October 2006 – February 2010: B.Sc. in Biology. University of Milan, Milan (Italy). Experimental work at University of Milan, section of Microbiology, Milan (Italy).

Thesis title: “Preliminary protocols to deep-sequencing for the identification of small RNAs in P. aeruginosa”.

Final Mark: 93/110; discussion date: February 26, 2010.

Advisors: Giovanni Bertoni, PhD; Silvia Ferrara, PhD.

• September 2000- July 2005: High School Diploma. Virgilio High School, Milan, (Italy).

List of publications

RESEARCH ARTICLES

Brambilla Pisoni, G., Ruddock, L. W., Bulleid, N. J., Molinari, M. (2015) Division of labor among oxidoreductases: TMX1 preferentially acts on transmembrane polypeptides. Mol Biol Cell 26(19):3390-400. doi: 10.1091/mbc.E15-05-0321.

Noack J.†, Fumagalli F.†, Bergmann T. J.†, Cebollero E.†, Brambilla Pisoni G., Fasana E., Fregno I., Galli C., Loi M., Simonelli L., Varani L., D’Antuono R., Raimondi A., Schorr S., Wilson-Zbinden C., Peter M., Hoffmann K., Zimmermann R. and Molinari M.* Sec62 regulates ER-phagy during recovery from ER stress. Under submission

REVIEWS

Noack, J., Brambilla Pisoni, G., Molinari, M. (2014) Proteostasis: bad news and good news from the endoplasmic reticulum. Swiss Med Wkly. 144, w14001, doi:10.4414/smw.2014.14001.

Tannous, A., Brambilla Pisoni, G., Hebert, D. N., Molinari, M. (2015) N-linked sugar- regulated protein folding and quality control in the ER. Seminars in Cell and Developmental Biology. 41, 79-89, doi:10.1016/j.semcdb.2014.12.001.

Brambilla Pisoni, G. and Molinari, M. (2016) Five Questions (with their Answers) on ER- associated Degradation. Traffic

Acknowledgements

A special thank goes to Maurizio Molinari for giving me the opportunity to work in his lab. He is The Person who allowed me to reach this goal, stimulating my scientific curiosity and surprisingly, my artistic sense. More importantly, thanks for introducing me to the TMX family. I want to thank the members of my thesis committee: Prof. Markus Aebi, Prof. Paola Picotti and Prof. Roberto Sitia, for insightful comments and for taking time to evaluate my work as well as my PhD thesis. For insightful discussions and for sharing crucial reagents I want to acknowledge Ineke Braakmann, Neil Bulleid, Lars Ellgaard, Ari Helenius, Paolo Paganetti, Lloyd Ruddock and Billy Tsai. For technical and scientific help I want to thank Dr. Manfredo Quadroni. Furthermore, I want to thank Daniel Hebert for giving me an unexpected feedback. For her incredible patience I want to thank Judith Zingg! For all the rest, I would like to thank the Molinari lab members: Carmela: thanks for your kind supervision in the lab. And many thanks for the flowers! Tatiana thanks for your precious help in and out of the lab, and, above all, thanks for cigarettes! Elisa: thanks for introducing me to the lab, for your friendship and for your 1000 words per second! Jessica and Julia: thank you girls, it has been a pleasure to have you as spiritual leaders! Timothy: thanks for your infinite repertoire of ... scientific stuff! Fiorenza: thanks for your dark side! Ilaria: thanks for choosing us for your Master, for being my junior PhD and for everything you did! and Marisa: … welcome to the jungle!