D o c t o r a l T h e s i s i n B i o c h e m i s t r y , S t o c k h o l m U n i v e r s i t y S w e d e n

Integration and topology of membrane proteins related to

diseases

P a t r i c i a L a r a V a s q u e z

Cover illustration “Lipids and membrane proteins in the membrane”. The lipids are represented as dancers and the membrane proteins as colourful boxes. ©Patricia Lara Vasquez, Stockholm University 2015 ISBN 978-91-7649-094-5 Printed in Sweden by Publit Sweden AB, Stockholm 2015 Distributor: Department of Biochemistry and Biophysics, Stockholm University

Integration and topology of membrane proteins related to diseases

Patricia Lara Vasquez

A mis padres, hermanos y sobrinos

List of publications

I. Wanngren J*, Lara P*, Öjemalm K, Maioli S, Moradi N, Chen L,

Tjernberg LO, Lundkvist J, Nilsson I, Karlström H. (2014) Changed

membrane integration and catalytic site conformation are two mech-

anisms behind the increased Ab42/Ab40 ratio by presenilin 1 familial

Alzheimer-linked mutations.

FEBS Open Bio 4:393-406

II. Ostuni A*, Lara P*, Armentano MF, Miglionico R, Salvia AM,

Mönnich M, Carmosino M, Lasorsa FM, Monné M, Nilsson I, Bis-

saccia F. (2013) The hepatitis B x antigen anti-apoptotic effector

URG7 is localized to the endoplasmic reticulum membrane.

FEBS lett. 587(18):3058-62

III. Saenz A, Presto J*, Lara P*, Akinyl OL, Nilsson I, Johansson J, Ca-

sals C. Folding and intramembraneous BRICHOS binding of the

proSP-C transmembrane segment.

Submitted

IV. Lee H*, Lara P*, Ostuni A, Presto J, Johansson J, Nilsson I, Kim H.

(2014) Live-cell topology assessment of URG7, MRP6102 and SP-C us-

ing glycosylatable green fluorescent protein in mammalian cells.

Biochem. Biophys. Res. Commun. 450(4):1587-92

* These authors contributed equally

Additional publications

V. Gadalla SE, Öjemalm K, Lara P, Nilsson I, Ericsson C, Zhao J,

Nistér M. (2013) EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells Biochem Biophys Res Commun 439(2): 203-208

VI. Goel, S, Palmkvist M, Moll K, Joannin N, Lara P, Akhouri R, Mo-radi N, Öjemalm K, Westman M, Angeletti D, Kjellin H, Lehtiö J, Hult AK, Olsson ML, von Heijne G, Nilsson I, and Wahlgren M RIFINs are Adhesins Implicated in Severe Plasmodium falciparum Malaria Nature Medicine just accepted

VII. Nilsson I, Lara P, Hessa T, Johnson AE, von Heijne G, Kara-

myshev AL. The code for directing proteins for translocation across ER membrane: SRP cotranslationally recognizes specific features of a signal sequence. J Mol Biol in press

Abstract

Membranes are boundaries that separate the cell from the external environ-

ment. Membrane proteins can function as e.g. receptors and channels, al-

lowing cells to communicate with the exterior and molecules to pass through

the membrane. The biogenesis of membrane proteins involves a protein-

conducting channel that aids the hydrophobic segments to partition into the

membrane and translocate the hydrophilic loops. Membrane proteins need to

fold to its native conformation including post-translational modifications and

assembly with other proteins and/or cofactors. If this regulated pathway goes

wrong the degradation machinery degrades the protein. If the system is fail-

ing can result in serious disorders. The main focus in this thesis is membrane

proteins associated to diseases.

We have studied mutations in the gene of presenilin 1, which is involved in

Alzheimer’s disease. We found that some mutations affect the structure and

other the function of the PS1. URG7 is an unknown protein associated with

liver cancer. We suggest it is localized and targeted to the ER membrane,

having an NoutCin topology. SP-C is important for our lungs to function. Mu-

tations can cause the protein to aggregate. We have studied the highly Val-

rich transmembrane segment (poly-Val) and its analogue (poly-Leu) and

show that poly-Leu folds into a more compact conformation than poly-Val.

We show that the C-terminal chaperon-like BRICHOS domain interacts with

the ER membrane, suggesting an involvement in poly-Val folding. We have

also confirmed the topology of URG7, MRP6 and SP-C poly-Val/Leu using

gGFP that is fused to the C-terminal of the protein.

Populärvetenskaplig sammanfattning

En cell är den minsta byggstenen i alla levande organismer. Människor består av

flera miljarder celler medan t.ex. en bakterie bara består av en enda cell. Cellen

innehåller arvsmassan, DNA:t som kodar för proteiner. Proteiner har livsvikta upp-

gifter i cellen, man kan säga att proteiner är cellens maskineri. Cellen är omslutet av

ett membran som består av lipider (fett) och membranproteiner som skiljer cellens

inre från yttre miljön. Membranproteiner har flera olika funktioner, som t.ex. kom-

municera med omvärlden och tillåta molekyler passera genom membranet som an-

nars är hel impermeabel.

För att membranproteiner ska kunna utföra sina uppgifter behöver de anta sin kor-

rekta struktur. Processen från att proteinet tillverkas till att den är funktionsduglig är

väldig komplex och kan ibland gå fel. Cellen kan snabbt reparera alla misstag, där-

emot om systemet inte fungerar som det ska, kan defekta proteiner uppstå och or-

saka sjukdomar. Denna avhandling handlar om membranproteiner och deras roll i

sjukdomar.

Vi har undersökt mutationer, som är kopplade till den ärftliga, så kallad familjära

Alzheimers sjukdom, som har en åldersmässigt tidig debut. Mutationerna i genen

som kodar för presenilin 1 (PS1) kan påverka strukturen och även aktiviteten av

PS1. URG7 är ett okänt protein, som är kopplad till levercancer. Vi har visat att det

är ett membranprotein, som sitter i endoplasmatiska nätverket. Surfactant C (SP-C)

är ett protein som kan aggregera, vilket resulterar i lungsjukdomar. Vi har undersökt

den transmembrana regionen av SP-C, som visar sig behöva hjälp av en annan do-

män i proteinet, den så kallade BRICHOS, för att anta sin korrekta struktur. Hur

detta sker behövs undersöka ytterligare, men vi visar att BRICHOS domänen kan

interagera med membran lipiderna. Vi har även bekräftat topologin av URG7 och

SP-C genom att använda oss av en metod som kan glykosylera ett grönt fluoresce-

rande protein i celler.

Resumen en español

Voy a tratar de explicar con lo que he trabajado durante estos 5 años de doctorado en

español, para que mis seres queridos puedan entender aunque sea un poquito mas:

Una célula es la parte más pequeña de la que están formados todo ser vivo. El nume-

ro de células puede variar, nosotros los humanos tenemos mas de billones células, en

cambio la bacteria solo tiene una. Todas las células están rodeadas de una envoltura,

una membrana de fosfolípidos y proteínas, que mantiene el medio interno diferen-

ciado del medio externo. Las proteínas de membrana se encargan de comunicarse

con el exterior. Además pueden funcionar como canales o transportadoras, permi-

tiendo que sustancias pueden entrar o salir de la célula. Para que las proteínas de

membrana puedan funcionar correctamente necesitan adoptar su estructura especifi-

ca. Este proceso, en la cual la proteína es sintetizada hasta que adopte su estructura

en la membrana, es enormemente complejo y en ocasiones puede fallar. Sin embar-

go, la celula esta preparada y puede rápidamente arreglar la falla. En cambio si el

sistema no esta funcionando como debe, puede resultar en enfermedades. El objetivo

de la tesis es estudiar proteínas que están relacionadas con enfermedades.

La enfermedad de Alzheimer es la forma más común de demencia. Mutaciones en el

gene de la proteína presenilin 1 (PS1) puede resultar en una forma mas agresiva de

Alzheimer, que aparece a menor edad. Hemos estudiado las mutaciones y pudimos

ver que afectan la estructura y la actividad de PS1. URG7 es una proteína descono-

cida que esta asociada a cáncer al hígado. Hemos demostrado que es una proteína de

membrana y esta localizada en el retículu endoplasmático. Surfactant C (SP-C) es

una proteína que se puede agregar resultando en enfermedades en el pulmón. Hemos

estudiado la transmembrana de SP-C y el carboxilo terminal, que contiene un domi-

nio, BRICHOS, que ayuda la proteína a adoptar su estructura. Hemos confirmado la

topología de URG7 y SP-C usando un método donde una proteína verde fluorescen-

te es glicosilada.

Contents

List of publications ..................................................................................... vi

Additional publications ............................................................................. vii

Abstract ...................................................................................................... viii

Populärvetenskaplig sammanfattning .................................................... ix

Resumen en español .................................................................................. x

Contents ....................................................................................................... xi

Abbreviations ............................................................................................ 14

Amino acids ............................................................................................ 15

1. Introduction .......................................................................................... 16

2. Biological membranes ........................................................................ 18

2.1 General features of biological membranes .............................. 18

2.2 Lipids in the membrane ............................................................... 19

2.3 The endoplasmic reticulum ......................................................... 22

3. Membrane proteins ............................................................................. 23

3.1 Membrane protein architecture .................................................. 24

3.2 General structural features of membrane proteins ................ 25

3.2.1 Hydrophobic mismatch ......................................................... 25

3.3 Properties of amino acids in membrane proteins ................... 26

3.3.1 Polar amino acids in transmembrane segments ............. 26

3.3.2 Proline and glycine in transmembrane segments ........... 27

3.3.3 Aromatic belt .......................................................................... 28

3.3.4 Snorkeling ............................................................................... 28

4. Biogenesis of membrane proteins ................................................... 29

4.1 The co-translational translocation ............................................. 29

4.2 The post-translational translocation .......................................... 31

4.3 The Sec translocon ....................................................................... 32

4.4 Accessory protein components .................................................. 34

5. Insertion of membrane proteins into the endoplasmic reticulum membrane ................................................................................................. 37

5.1 Topology of membrane proteins ................................................. 38

5.2 What factors determines the topology of membrane proteins? ..................................................................................................................... 38

5.2.1 Positive inside rule .................................................................. 40

5.2.2 Hydrophobicity ......................................................................... 40

5.2.3 Transmembrane lenght .......................................................... 41

6. Diseases related to proteins studied ............................................... 42

6.1 Alzheimer’s disease ........................................................................ 42

6.2 Interstitial lung disease ................................................................ 43

6.3 Hepatocellular carcinoma .............................................................. 44

7. Methodology ......................................................................................... 45

7.1 The model protein: Leader peptidase ........................................ 45

7.1.1 Lep H2 and Lep H3 .................................................................. 46

7.2 Determining the propensity to integrate into the ER membrane ................................................................................................. 47

7.3 Determining the topology of membrane proteins ................... 47

7.3.1 Topology prediction ................................................................ 48

7.3.2 Topology mapping .................................................................. 49

7.3.2.1 Glycosylation assay ......................................................... 49

7.3.2.2 Protease protection assay .............................................. 50

7.2.3.3 Glycosylatable GFP assay ............................................... 50

8. Summary of papers ............................................................................ 51

9. Conclusions and perspectives ........................................................... 54

Acknowledgements .................................................................................. 57

References ................................................................................................. 61

Abbreviations

Aβ amyloid β-peptide AD Alzheimer’s disease ER endoplasmic reticulum FAD familial AD GFP green fluorescent protein i-CLiPs intramembrane cleaving proteases ILD interstitial lung disease Lep leader peptidase MGD minimal glycosylation distance OST oligosaccharyltransferase PA phosphatidic acid PC Phosphateidylcholine PE phosphateidylethanol PI phosphateidylinositol PM plasma membrane PS phosphateidylserine PS1 Presenilin 1 RNC ribosome-nascent chain SM sphingomyelin SP signal peptidase SR SRP receptor SRP signal recognition particle TM transmembrane

Amino acids Ala Alanine (A)

Arg Arginine (R)

Asn Asparagine (N)

Asp Aspartic acid (D)

Cys Cysteine (C)

Gln Glutamine (Q)

Glu Glutamic acid (E)

Gly Glycine (G)

His Histidine (H)

Ile Isoleucine (I)

Leu Leucine (L)

Lys Lysine (K)

Met Methionine (M)

Phe Phenylalanine (F)

Pro Proline (P)

Ser Serine (S)

Thr Threonine (T)

Trp Tryptophan (W)

Tyr Tyrosine (Y)

Val Valine (V)

16

1. Introduction

All living organisms visible or non-visible are made of cells. The cell is the

basic unit of life and the smallest one that is capable to independently repro-

duce itself. It can vary in size and shape and is bounded by a cell membrane.

The membrane is composed of lipids and proteins that build up a hydropho-

bic barrier that separates the cell contents from the external environment. It

is important for the cell to take up nutrients and other molecules, as well as

excrete waste products to its surroundings in order to function, and for that

reason the membrane cannot be completely impermeable. Specialized pro-

teins embedded in the membrane, called membrane proteins serve to

transport specific molecules from one side to the other.

There are different types of cells, which fall in the three distinct domains of

life. Two large groups, archaea that inhabit extreme environments and pro-

karyotes (bacteria) are microorganisms that do not have a nucleus (Fig. 1A).

Cells with a membrane bounded nucleus make up the third group, called

eukaryotes e.g. animal (Fig. 1B) and plant cells. Eukaryotes have besides a

nucleus, other compartments, called organelles that are surrounded by mem-

branes, such as the endoplasmic reticulum (ER), mitochondria, chloroplasts,

peroxisomes, Golgi and lysosome, each with specific functions. Although,

there are many differences between archaea, prokaryotes and eukaryotes,

they share common features, one is the storage of their hereditary infor-

mation in form of DNA. The DNA carries instructions for the production of

the cell machinery.

The main focus of this thesis is the study of eukaryotic membrane proteins,

how they insert into the ER membrane and the understanding of membrane-

protein topology that reveals how they sit in the membrane. Interestingly, the

17

specific proteins studied are connected to diseases, the first is involved in

Alzheimer’s disease, the second in liver cancer and the third is crucial for

our lungs and breathing.

A

B

Fig. 1. Schematic illustration of a prokaryot cell (A) and an animal eukaryotic cell

(B).

18

2. Biological membranes

2.1 General features of biological membranes Membranes are the boundaries that surround the cell and maintain the cell

integrity by separating the inside milieu from its exterior environment. The

two major components are lipids and proteins. These molecules are charac-

terized by being amphiphilic, a property that constitutes the structure of the

membrane in an aqueous solution. The lipids are organized in a double layer,

which thermodynamically favors and stabilize the membrane by the interac-

tion of hydrophobic acyl chains facing each other in the interior of the mem-

brane excluding the contact with water. On the other hand, the polar head

groups are oriented to the outer, aqueous space. Our understanding and view

of the dynamics and structure of the biological membrane is based on the

‘fluid mosaic model’ presented by Singer and Nicholson in 19721. The

membrane is seen as a two-dimensional liquid where lipids and proteins are

in constant motion, freely moving laterally within the membrane matrix (Fig.

2A). However, this model has been updated as seen in Fig. 2A, few integral

proteins floating in the fluid bilayer lipid phase leaving most of the lipids

unperturbed. New experimental approaches in the membrane field sustain a

more crowded lipid bilayer with high numbers of integral membrane pro-

teins limiting the free lateral diffusion of the molecules (Fig. 2B)2; 3. Moreo-

ver, the surface of the bilayer is often crowded with peripheral proteins that

sometimes can be transiently attached. Oligosaccharides, sugar molecules

also protrude from the membrane on the cell surface and play important

19

roles in cell-to-cell recognition and signaling. They are often attached cova-

lently to lipids or proteins.

A B

Fig. 2. Membrane models. Model structure of the cell membrane based on the Sing-

er Nicolson model. Lipids and proteins are freely diffusing laterally1 (A). An update

version of a more crowded membrane according to Engelman2 (B).

2.2 Lipids in the membrane As mentioned above, membrane lipids are considered as amphipathic struc-

tures, they have a hydrophobic tail and polar head region (Fig 3). In an

aqueous solution they spontaneously form a bilayer structure, shielding their

hydrophobic moieties, facing them to each other and their polar structures

are exposed towards the water environment. This ‘hydrophobic effect’ is the

foundation of the biological membrane structure. The hydrophilic head

group determines the charge of the lipid. They can be neutral, zwitterionic,

with a positive and negative dipole, resulting in no overall charge, or they

can be negatively charged.

The three major kinds of lipids in a biological membrane are phospholipids,

glycolipids and cholesterol. Considering the properties of the two first lipids,

such as the diversity of chain length of the fatty acids, and the varying de-

20

gree of saturation, including the different head groups that exist, one can

come up to a high number of different lipid species. There are more than

hundred different lipid species present in a membrane of a prokaryotic cell

such as Escherichia coli4, and more than 1 000 different types in a eukaryot-

ic cell5. The features of a lipid contribute to its shape, so lipids can be divid-

ed into three groups based on their structure: cylindrical, conical, and invert-

ed conical, which display a no curvature, negative curvature respectively

positive curvature nature (Fig. 3), when they are accumulated locally.

A

B

Fig. 3. Membrane lipids. Schematic structure of a phospholipid, in this special case,

phosphateidylcholine (A). The polar head group and the hydrophobic tail can vary in

size resulting in different lipid shapes, such as cylindrical, conical and inverted coni-

cal. Adapted from Suetsugu et al.6 (B).

21

Phosphateidylcholine (PC), phosphateidylethanol (PE), phosphateidylserine

(PS) and sphingomyelin (SM) are the most common lipids in the eukaryotic

plasma membrane (PM). Both PC and PS are cylindrical lipids that form flat

bilayers, while PE contributes with a negative curvature structure. PS pos-

sesses a negatively charge head group. Additionally, two other lipids also

exist but to lower extent, that contribute to negative charges, they are phos-

phatidic acid (PA) and phosphatidylinositol (PI) as well as, its phosphory-

lated derivatives, phosphoinositides6. A special type of lipid can be associat-

ed to a certain type of organelles. For example, cardiolipin is mainly found

in the inner mitochondrial membrane, while the PM is enriched in sphin-

golipids and sterols7; 8.

Membranes are asymmetric; the lipids are asymmetrically distributed in the

inner and outer leaflet of the lipid bilayer9, with this, the membrane can

adopt different shapes. Lipids that induce curvature (PE, PA, and PI), con-

tributing to membrane bending, are important for vesicle budding, fusion

and fission. Moreover, the asymmetry has essential regulatory features, for

example, PS is present exclusively in the inner leaflet of the PM. However,

when PS is transported and exposed to the cell surface, in the outer leaflet, it

is a signal for apoptosis10.

Furthermore, the phospholipid composition varies between cell types and

membranes. The eukaryotic PM is enriched in sphingolipids and cholesterol.

The first has saturated tails and the second has properties that allow it to

intercalate between the lipids, generating a more tightly packed membrane8;

9, which is characteristic for the PM in contrast to the intracellular organelle

membranes. The lipid composition of the PM, including the presence of

more saturated PS and PE in yeast for example11, contributes to a more rigid

and stable membrane. One can suggest that this is the case for other cell

types as well, as the general view of the PM is that it is tightly packed, high-

22

ly charged (high PS and phosphoinositides content)12 and more lipid-

asymmetric than other membranes9, which defines its function as the perme-

ability barrier of cells.

2.3 The endoplasmic reticulum The eukaryotic organelle endoplasmic reticulum (ER) is highly dynamic and

its membrane forms a network of tubules and cisternae that separates its

interior, the lumen, from the cytoplasm13. In addition to being the major site

of lipid synthesis, it is also the platform for protein production, folding and

quality control. Although cholesterol is synthesized in the ER, the ER mem-

brane displays low levels of cholesterol and also of sphingolipids. The level

of cholesterol is maintained low since it is transported to other organelles.

This is possible because the ER is in contact with the nucleus, mitochondria,

peroxisomes, and the PM in the cell8; 13. The scarcity of sphingolipids and

cholesterol, including the abundance of unsaturated fatty acids, results in

looser lipid packing that is consistent with the role of this organelle in trans-

porting and inserting newly synthesized lipids and proteins into the lipid

bilayer8. In addition it has been suggested that the ER is less asymmetric

than the PM and may be regulated by the presence of ATP-dependent lipid

transporters, they so called flippases, floppases and scramblases9. The first

one move phospholipids from the outer to cytosolic leaflet, the second

moves phospholipids from the cytosolic to the outer leaflet and the third

moves lipids in either direction toward equilibrium. In summary, ER is char-

acterized by loose lipid packing and seems to be less asymmetric and elec-

trostatic, due to the weakly charged cytosolic leaflet, because of the low

content of negatively charged lipids12.

So far I have only mentioned one of the major components in the biological

membrane, the lipids, however, in the next chapter I will explain more about

the membrane proteins and their role in the lipid bilayer.

23

3. Membrane proteins

Membrane proteins are important molecules that have crucial and specific

roles, and are involved in many biological functions. They make the cell

membrane permeable to molecules needed for the cell to function and they

allow the cell to communicate with its environment. In addition to maintain-

ing the shape of the lipid bilayer, they act as sensors (receptors) of external

signals allowing the information to cross the membrane and for the cell to

respond to them. Furthermore, they function as transporters for polar mole-

cules such as ions, water and peptides and as channels, allowing molecules

to pass the membrane by diffusion14; 15. Not to forget, they are important

factors of transforming energy, converting chemical energy into electrical

and synthesizing ATP, the cellular energy currency of life16.

There are three types of membrane proteins. Peripheral (or extrinsic) mem-

brane proteins interact weakly and reversible with the membrane on its sur-

face. They are distinguished by how they can be released from the mem-

brane, by using relatively gentle treatments such as high salt or high pH

without affecting the membrane structure. Peripheral proteins associate with

the membrane through electrostatic interactions and hydrogen bonding with

the polar head group of the lipids and hydrophilic domains of proteins1; 3.

The second group, amphitropic proteins are found in the interior of the cell,

in the cytosol. They can either interact non-covalently with lipids and pro-

teins or they can have one or more lipids covalently attached to them.

24

The third group of membrane proteins is critical for structural integrity of

membranes. Integral (or intrinsic) membrane proteins are buried inside the

bilayer, surrounded by lipids, with their hydrophilic domains protruding into

the aqueous milieu. They are strongly associated with the membrane and

require harsher treatment such as detergents, organic solvent, or denaturants

to remove them from the membrane.

3.1 Membrane protein architecture Determination of the three-dimensional (3D) structures of membrane pro-

teins has revealed two types of structural motifs that dominate in membrane

proteins, α-helical bundles and β-barrels16; 17; 18. The latter one found almost

exclusively in the outer membranes of Gram-negative bacteria and in mito-

chondria and chloroplast of eukaryotes, while most proteins in cellular

membranes are bundles of α-helices. The folding into these two basic sec-

ondary structures (Fig. 4) is favorable for a protein embedded in a non-

aqueous environment, which are stabilized by having the hydrogen bonding

of the polar backbones satisfied14.

A B

Fig. 4. Structures of the two major types of membrane proteins. The OmpA β-barrel

protein (PDB: 1QJP)19 (A). The Rhodopsin α-helical protein (PDB: 2I35) (B).

25

Three years after the Singer and Nicholson model of the cell membrane was

published, the 3D structure of the first integral membrane protein was re-

solved, the light driven proton pump of bacteriorhodopsin from Halobacte-

rium halobium20. Even though, the resolution was not so high, it revealed for

the first time that membrane proteins consist of α-helical transmembrane

(TM) segments oriented perpendicular to the plane of the membrane (Fig 5).

Years after came the first high-resolution crystal structure of the bacterial

photosynthetic reaction center21 and after that until now the number of de-

termined structures has been increasing exponentially14; 22. However, it has

been, and still is a challenge to work with membrane proteins, as they are

unstable outside the membrane environment. In order to stabilize them they

need to be reinserted into a kind of lipid, detergent milieu, by adding ligands

or inhibitors, or by introducing point mutations to generate a more intrinsi-

cally stable protein16.

3.2 General structural features of membrane proteins The high-resolution membrane protein structures have revealed important

general structural features: The length of a typical α-helix is between 20 and

30 amino acids. The hydrocarbon core of the membrane is approximately 30

Å, and the membrane-water interface around 15 Å. To fit a TM helix in the

core would require around 20 amino acids. Interactions between the TM

segment and lipids are essential to many cellular activities.

3.2.1 Hydrophobic mismatch Mismatch can occur in which the hydrophobic length of the TM segment is

longer than the core (positive mismatch) or the opposite, the core is thicker

than the TM (negative mismatch). There is a variety of ways to minimize the

energetically unfavorable exposure of hydrophobic groups to the hydrophilic

environment and polar groups of the peptide to the lipid bilayer23. Studies

26

have indicated that both TM segments and lipids respond to mismatch. The

length of a TM segment can vary a lot, when it is longer than the thickness

of the membrane it tilts with respect to the bilayer or results in kinked and

even interrupted α-helices24; 25. Alternatively, it can oligomerize or even

aggregate to minimize the exposed hydrophobic parts. In contrast, when the

hydrophobic part is too small it can result again in aggregation or changes in

the side chain orientation. Furthermore, as a consequence ‘snorkerling’ could

occur, discussed below. Although, it has been experimentally shown that a

TM segment as short as 10 residues can be inserted into the ER membrane26,

too short TMs may adopt a surface localization27. Lipids are also capable of

responding to hydrophobic mismatch by stretching or disordering their fatty

acid chains. Moreover, while the thickness of the membrane can vary with

the lipid composition, the plasma membrane is likely to be thicker than the

ER due to the high content of cholesterol and sphingomyelin. This suggests

that hydrophobic mismatch may be required for specific functions in the

membrane, such as affecting protein sorting to particular organelles27; 28 and

seems to be involved in the functionality of several proteins29; 30.

3.3 Properties of amino acids in membrane proteins Another feature that membrane proteins share, is the amino acid distribution,

which shows preferential locations within the TM helix24; 31. Not surprising-

ly, the hydrophobic residues such as Leu, Ile, Val. Phe and Ala are the most

frequent ones in the TM helices buried in the hydrocarbon core.

3.3.1 Polar amino acids in transmembrane segments As expected polar and charged residues are less frequently in a TM segment.

If present, they are conserved and often involved in important biologically

functions, conformational specificity and thermodynamic stability14; 24; 32.

Nevertheless, the high energetic cost can be compensated by the formation

27

of salt bridges, which can drive specific interactions between helical seg-

ments33 and even facilitate hairpin formation34. The helix-helix interaction

has been widely studied35, their associations being governed by already men-

tioned, electrostatic and also van der Waals interactions. For example, Asp-

Asp or Asn-Asn interactions stabilize the TM topography of helices in the

bilayer36.

3.3.2 Proline and glycine in transmembrane segments Despite the fact that Gly and Pro are known as ‘helix-breakers’ they are

found in TM helices, Gly is especially crucial in helix-helix interactions37.

The dimeric protein, Glycophorin A (GpA) has been well studied and re-

veals the involvement of Gly in dimerization of membrane proteins37; 38. The

many studies of GpA led to the identification of the GxxxG motif found in

several membrane proteins39. The small side chain of Gly allows a very close

contact between two helices. Pro is unique because its side chain lacks the

proton necessary for hydrogen bonding, which results in a kink in the protein

backbone40. However, the structure of Rhodopsin shows that a Pro in the

middle of a TM helix does not necessary always cause bending41. Pro has

also been suggested to be involved in helix-helix interactions of many mem-

brane proteins40.

Fig. 5. Model of the first 3D structure of the bacteriorhodopsin membrane protein

obtained by electron microscopy at 7 Å resolution20.

28

3.3.3 ‘Aromatic belt’ The aromatic amino acids Tyr and Trp are enriched at the ends of TM heli-

ces, in the lipid-water interface. Their amphipathic character allows them to

hydrogen bond to lipid head groups. One possible explanation is that it could

provide extra stability to the helix, serving as an anchor. Furthermore, it has

been shown that both Tyr and Trp destabilize the TM state when it is in the

central part of a TM helix. On the other hand, Phe, also aromatic and fully

hydrophobic shows a preference for the hydrocarbon core rather than for the

interface space17; 24; 42; 43; 44.

3.3.4 ‘Snorkeling’ Charged residues are highly costly to keep within the membrane core. Inter-

estingly, both Lys and Arg are more tolerated at the edges of the TM helix.

Probably, because their long, flexible alipathic side chain buried inside the

bilayer could ‘snorkel’ up into the head group region, so that the positively

charged end may interact with the phosphate groups. Nevertheless, negative-

ly charged residues have a small side chain and might be repelled by the

negatively charged head groups, which would be energetically unfavorable

to keep in the interface43; 44; 45.

29

4. Biogenesis of membrane proteins

How do membrane proteins end up in the lipid bilayer? First, they are syn-

thesized by the ribosome, which translates the mRNA into amino acids and

catalyses the peptide bond formation. The growing polypeptide chain is re-

leased into the cytosol, where the ribosome operates. However, the hydro-

phobic nature of a membrane protein would cause the protein to aggregate in

the polar milieu of the cytosol. To overcome this problem the nonpolar parts

of the protein needs to be masked before entering the membrane, facilitated

by the translocon, in order to avoid unfavourable contacts with the polar

environment. There is a common evolutionarily conserved pathway for most

membrane and secreted proteins, where the ribosome and the translocon

work together with the signal recognition particle (SRP). Most of eukaryotic

membrane proteins insert co-translationally, fold and oligomerize in the ER

membrane. Moreover, those that are destined to function in other membrane

compartments such as the plasma membrane are transported in vesicles to

their target destination46; 47.

4.1 The co-translational translocation The SRP-dependent pathway starts when a signal sequence or the first TM

of the growing nascent chain emerges from the ribosome and is recognized

by the SRP. The cleavable signal sequence has a positively charged N-

terminal region followed by a hydrophobic domain composed of 7-15 resi-

dues and a polar C-terminal region. In contrast, the non-cleavable hydropho-

bic domains that anchors the protein in the membrane are longer, usually 19-

27 residues48; 49. When SRP binds to the signal sequence it pauses the nas-

30

cent chain elongation50; 51 and brings the whole ribosome-nascent chain

(RNC) complex to the ER membrane by interacting with the SRP receptor

(SR)52. Both SRP and SR have a GTPase domain and their contact and inter-

action occurs through the GTPase modules. They are bound to GTP when

they are associated with the RNC, however, when GTP is hydrolyzed the

whole complex is disassembled and SRP and SR are recycled for the next

run52. Moreover, it promotes ribosome binding to cytosolic loops of the pro-

tein conduction channel, the translocon. The docked ribosome can reinitiate

the elongation of protein synthesis, while it releases the growing polypeptide

into the proteinous environment of the translocon, which allows soluble do-

mains to cross the membrane and hydrophobic TM segments to exit laterally

into the lipid phase53 (Fig. 6).

Fig. 6. Overview of the co-translational SRP-depedent targeting pathway of newly

synthesized polypeptides54.

The RNC targeting is universally conserved over all domains of life. The

SRP pathway mechanism is similar between eukaryotic and prokaryotic

systems, although there are small differences. In prokaryotes the RNC is

31

destined to the bacteria inner membrane (IM). The bacterial SRP contains

the conserved protein component Ffh (called SRP54 in eukaryotes) and the

4.5S SRP RNA. They are mainly involved in the targeting of inner mem-

brane proteins to the translocon, while secreted proteins utilize the post-

translational pathway55; 56. The bacterial SRP lacks the subunits that are as-

sociated with the translational arrest seen in eukaryotes. Moreover, the bac-

terial SR, called FtsY, is a single protein that lacks TM segments and may

interact weakly with the bacterial IM55.

4.2 The post-translational translocation In the post-translational pathway, the protein is completely synthesize in the

cytosol and thereafter targeted and inserted into the ER membrane. It begins

with the binding of cytosolic chaperones while the nascent chain emerges

from the ribosome in order to prevent premature folding before the polypep-

tide is transported through the channel. The substrate is later targeted to the

Sec61 complex, which interacts with the Sec62/Sec63 complex. The chaper-

one releases the nascent chain while it is translocated through the channel46;

57. On the lumenal side of the ER the chaperon BiP binds to the polypeptide

preventing the polypeptide chain to slide back to the cytosol and instead

allowing it to move forward46. BiP is an ATPase that provides energy for

translocation58. The mechanism is the following: BiP in its ATP-bound state

interacts with Sec63, when ATP is hydrolyzed, BiP binds to the polypeptide

chain in its ADP state. When the nascent chain has moved further in the

forward direction, the next BiP molecule can bind and so on. Prokaryotes

have instead a cytosolic ATPase, SecA that aid long hydrophilic domains

and fully synthesized polypeptides to cross the ER membrane trough the

translocon46. Although, archaea lack both BiP and SecA, it has been suggest-

ed that posttranslational translocation may occur as well but how it is ener-

gized remains unclear55.

32

4.3 The Sec translocon The universal protein-conducting channel is found in all kingdoms of life,

called Sec61 in eukaryotes and SecYEG in bacteria and archaea. The Sec

translocon allows membrane proteins and secretory proteins to translocate

and integrate into the ER membrane59. The eukaryotic Sec61 channel com-

plex consists of three subunits, α, β and γ, both the α- and the γ-subunits are

very well conserved and essential for cell viability. In 2004 van den Berg et

al. determined the x-ray structure of the archaea Sec complex from Meth-

anococcus jannaschii60 and after that additional crystal structures have been

resolved with distinctive conformations61; 62. Despite the evolutionary dis-

tance between the different organisms, superimposing the structures indi-

cates a high degree of similarity reflecting how well conserved it is63 in both

prokaryotes and eukaryotes64; 65.

Fig. 7. Crystal structure of the SecY complex from M. jannaschii (PBD: 1RHZ).

View from the side (left) and from the top of the cytosolic side (right). The SecY-

subunit (α-subunit) contains 10 TMs (in grey). The lateral gate is formed by TM2a

(orange) and TM7 (blue) that allows hydrophobic segments to partition into the

membrane. The β-subunit and γ-subunit are depicted in black. The blue lines indi-

cate the approximate boundaries of the membrane

33

The 10 TM segments of Sec61α (SecY) subunit forms the pore, which has

the form of an hourglass viewed from the side, with a constriction ring com-

posed of hydrophobic residues in the cytoplasmic half of the membrane.

However, studies have indicated that the pore has an aqueous environment66;

67; 68. The α-subunit is divided into two halves (TM1-5 and TM6-10) that

forms a ‘clam shell’ and has an interconnected hinge at the cytoplasmic loop

between TM5 and TM6. Furthermore, from the lumenal side a short helix,

TM2a also referred to as the ‘plug’ fills the center of the cavity. It maintains

the channel impermeable to ions and small molecules, and it was suggested

to be removed when the polypeptide enters, allowing translocation (Fig. 7).

However, Gogala et al. reported that when the nascent chain is translocated

the plug is only slightly displaced65. Taken together, it is important to main-

tain the membrane barrier, particularly for prokaryotes, since the proton gra-

dient across the membrane is central for ATP-production and some trans-

ports69; 70. Moreover, the ribosome on the cytosolic side and chaperones such

as BiP on the luminal side has been suggested to seal to the cytosol or lumen

respectively depending on where the nascent chain is directed71; 72. They may

also assist in the folding of the growing polypeptide chain73. The lateral gate

releases TM segments to partition into the hydrophobic environment of the

lipid phase. Although the mechanism is unknown it has been suggested that

the presence of a hydrophobic peptide stabilizes an open conformation74.

Photocross-linking experiments have shown that the signal sequence con-

tacts and intercalates between TM2 and TM775. Additionally, this has been

confirmed by the recent crystal structure of Sec61, where TM2 and TM7

move apart separating the two halves by approximately 12 Å in the presence

of a hydrophobic TM segment65.

The function of Secβ (SecG) is unknown, non-essential and has only minor

contacts with the periphery of the α-subunit. It has been postulated to have a

kinetic effect and to facilitate the cotranslational translocation60; 76. However,

34

Secγ (SecE) is essential and helps to maintain the structure of the complex

by holding together the two halves of the α-subunit mentioned above60.

4.4 Accessory protein components Reconstitution of protein translocation components into proteoliposomes

revealed that for efficient translocation only three proteins are necessary, the

Sec61 complex, SR and the translocating chain-associated membrane protein

(TRAM)77. However, for optimal and proper transport of varying substrates

in the secretory pathway it is likely that other proteins involved. There are

several additional membrane components in addition to the ones mentioned

that have been implicated in the translocation process78, and they seem to be

in a close proximity of the Sec6179.

The signal peptidase (SP) complex is the protease that removes the N-

terminal signal sequence from secreted proteins and some membrane pro-

teins on the lumenal side of the membrane80. The crystal structure of the E.

coli signal peptidase has revealed that it is a serine endoprotease81. SP has a

substrate specificity for small and uncharged residues, such as Ala82. Follow-

ing the removal of the signal sequence, the polypeptide is folded, modified,

and salt bridges are formed. However, the signal sequence left in the mem-

brane is further cleaved by a signal peptide peptidase (SPP). SPP belong to

the family of intramembrane cleaving proteases (i-CLiPs) including the pre-

senilin (PS) family of aspartyl proteases, the zinc metalloprotease site-2 pro-

tease family and the rhomboid family of serine proteases. SPP cleavage re-

quires helix-breaking residues within the TM region of the substrate, while

positive charges that flank the TM region have an inhibitory effect. Another

requirement is that SP must first cleave on the lumenal side preparing the

substrate for the subsequent intramembrane cleavage performed by SPP83.

The aspartic proteases, PS and SPP share identical active site motifs, GxGD

and the highly conserved PAL motifs, indicating that they have a common

35

catalytic mechanism84. However, their active site is positioned opposite

within the plane of the membrane. This difference is due to how their sub-

strates orient in the membrane, PS has substrates with an NoutCin topology,

whereas SPP has substrates displaying an NinCout topology85; 86. In particular-

ly PS is interesting for this thesis in its involvement in Alzheimer’s disease,

as discussed later.

N-linked glycosylation is one of the most common covalent protein modifi-

cations in mammals and it is essential for the folding of the protein and even

for oligomerization, quality control and transport87. It is s not only restricted

to eukaryotes, although it is less frequent in prokaryotes88. N-linked glyco-

sylation occurs co-translationally by the oligosaccharyltransferase (OST)

multi-subunit protein complex that is closely associated with the

translocon89; 90. It transfers the preassembled oligosaccharide,

Glc3Man9GlcNAc2 in higher eukaryotes, to a specific Asn residue in the

protein. The catalytic site is located on the lumenal side of the ER mem-

brane87.

TRAM spans the membrane eight times with the N- and C-terminal facing

the cytoplasm91 and is involved early in the translocation and insertion of

secreted and membrane proteins92. Moreover, it has been suggested to func-

tion as a chaperone for short, weak signal sequences and less hydrophobic

TM segments with charges93; 94.

TRAP is a tetrameric complex95. Although, its main function remains un-

clear, it has been proposed to facilitate the initiation of substrate transport96.

In addition, it might affect the topology of membrane proteins, promoting C-

terminal translocation for those that lack topogenic signals, stabilizing an

NinCout orientation97.

36

In addition, it is worth to mention the ribosome-associated membrane protein

4 (RAMP4), a small single-spanning membrane protein93; 98. It seems to be

involved in folding99, regulating N-linked glycosylation of proteins100 and

stabilizing membranes proteins in response to stress101. Moreover, PAT-

10102, and BAP31103 have also been reported to interact with the translocon.

37

5. Insertion of membrane proteins into the endoplasmic reticulum membrane

When the membrane protein has been effectively co- or post-translational

targeted to the translocon, it needs to be recognized and inserted into the ER

membrane. How this occurs is not completely understood although many

studies indicate that the major factor is the overall hydrophobicity of the

helix104. A broad study where each of the 20 naturally amino-acid residues

were systematically tested across a model TM segment composed of Ala and

Leu residues led to the well established ‘biological scale’. These results pro-

vided comprehensive information regarding membrane integration and loca-

tion dependence of the individual residues in a TM helix105; 106. The experi-

mental data from the study has been used to develop and improve algorithms

for membrane topology prediction107. The ‘biological scale’ proposes that

the insertion is a thermodynamically driven equilibrium process in which the

partitioning into the lipid phase is dependent on the hydrophobicity of the

TM domain. The TM segment positioned in the polar channel may open the

lateral gate so it can access the hydrophobic milieu outside and diffuse into

the membrane94. However, the process can also be kinetically controlled,

where the Sec translocon plays a more active role than just providing a site

in the membrane where TMs equilibrate between lipid and aqueous phases74.

Substituting the hydrophobic residues in the pore ring of the yeast Sec61p

affected the efficiency of TM segment integration into the lipid phase108.

This suggests that the Sec translocon sets the hydrophobicity threshold for

membrane integration. Moreover, marginally hydrophobic TM segments are

more efficiently inserted when translocation is slower109.

38

5.1 Topology of membrane proteins During integration, the membrane protein needs to adopt the correct topolo-

gy and fold into a final compact native structure, which is important for

function. Membrane topology can be described as how many times a mem-

brane protein spans the lipid bilayer and where the soluble loops that connect

the TM segments are oriented relative to the plane of the membrane. This

information can help us to better understand the structure and the function by

revealing the localization of specific domains of a membrane protein. How

do membrane proteins achieve their topology? The simplest model suggests

that in a multi-spanning membrane protein, the first TM segment is the one

that determines the orientation of the subsequent ones. However, this is not

always the case, there are exceptions, in which the internal TM segments can

reorient and change the whole topology. One example is the SecG compo-

nent of the Sec translocon, which flips between different topologies during

translocation of the nascent chain110. Nevertheless, there are TM segments

that are not sufficiently hydrophobic to be co-translationally released into the

lipid bilayer, instead they are inserted post-translationally altering the orien-

tation of already inserted TM helices, as for aquaporin 1111. Controversially

of what has been described, there are dual-topology proteins. These proteins

can insert in two opposite orientations, and are fully functional when adopt-

ing both topologies, e.g. the well-studied EmrE. EmrE is a small multidrug

resistance homodimer protein that has been proposed to have two oppositely

orientated molecules, displaying a functional dual-topology protein112.

5.2 What factors determines the topology of a membrane

protein? Membrane proteins can adopt different types of topology that result in an

NoutCin or an NinCout orientation (Fig. 8) depending on various factors, such

39

as the length of the TM segment, the hydrophobicity, distribution of charged

residues and other features. Some TM segments, e.g. marginally hydropho-

bic TM helices are unable to integrate by themselves into the membrane and

need the interaction with adjacent more hydrophobic TM segments113; 114.

These poorly hydrophobic TMs are retained close to the Sec translocon by

polar interactions to pack and assembly properly before they are released

into the lipid bilayer115. This protein-protein interaction at the translocon has

been observed for several proteins102; 116; 117; 118. Furthermore, premature dis-

location of a mariginally hydrophobic TM into the membrane can cause

aggregation118.

Fig. 8. Different types membrane protein topologies

In membrane proteins, loops with positively charge residues tend to position

in the cytosol, the ‘positive-inside rule’ is a general mechanism found in all

organisms119. Crystal structures of membrane proteins have revealed a num-

ber of helices, ‘reentrant loops’120; 121; 122, that do not span the whole mem-

brane, but rather insert as hairpins that orient the N- and C-terminal end of

the TM on the same side of the membrane123; 124; 125. Another important factor

in membrane protein integration is the lipid composition of the lipid bilayer.

The topology of lactose permease (LacY) was largely altered when inserted

40

in a membrane lacking phosphatidylethanolamine, signifying the importance

of lipid influence on topology. Interestingly, by inducing PE synthesis post-

assembly of LacY, the TM domains could reorient to their native orientation,

indicating that the process is completely reversible126.

The cleavable signal sequence of the secreted proteins inserts into ER in an

NinCout orientation i.e. a type I membrane protein. How the N-terminal reori-

ents has been under debate, it has been proposed to either go in ‘head-first’

and then flip127; 128, or it inserts as a hairpin129. The N-terminus can be fixed

on the lumenal side of the ER membrane by introducing a glycosylation site

in the N-terminus, thus suggesting that the signal sequence can reorient with-

in the translocon130.

5.2.1 ‘Positive inside rule’ The phenomenon of having positive charges predominantly in the hydro-

philic loops on the cytoplasmic side of the membrane is well preserved131.

The positive inside rule displays a strong topogenic signal104; 132, as when

deleted or introduced the topology is changed132; 133. One explanation could

be that the positive charges are arrested in the cytosol134; 135; 136, probably

because they interact with the negatively charged lipid head groups137. Nev-

ertheless, the positive charges contribute to the insertion of TM helices into

the membrane more efficiently104; 138. In addition, the translocon may be

involved in the initial orientation of the TM by following the positive inside

rule; conserved charges at the Sec61p of yeast seem to interact with the nas-

cent chain providing the driving force for signal orientation139.

5.2.2 Hydrophobicity The more hydrophobic the TM segment is the more it tends to insert with an

NoutCin orientation independently of the charges of the flanking region127; 140.

An explanation is that very hydrophobic helices may rapidly exit the lateral

41

gate by interacting with the hydrophobic core of the lipid bilayer94. Never-

theless, the sliding model has been proposed, which suggests that strongly

hydrophobic TMs never enter the polar channel but instead slip into the lipid

bilayer along the lateral gate of the translocon141. In contrast, natural cleava-

ble signal sequences (NinCout) are less hydrophobic, and remain longer time

around the translocon, which allows them to reorient during translation.

5.2.3 Transmembrane length The length of the hydrophobic sequence can also determine the orientation

of the TM. Longer sequences show a preference towards an NoutCin topology,

localizing the N terminal in the lumen140; 142; 143. Once again, cleavable signal

sequences have shorter hydrophobic segments than TM segments adopting

an NinCout topology49, confirming the TM length as a determinant in defining

the orientation of the hydrophobic helix.

42

6. Diseases related to the proteins studied

Proteins are the most abundant biological macromolecules in all cells, and

have a variety of functions. It is difficult to imagine a world without pro-

teins, which are involved in perhaps every process occurring in the cell.

Membrane proteins constitute about 30% of all proteins. It is therefore not a

surprise that malfunctioning membrane proteins can cause a wide range of

diseases. Here is a presentation of the diseases that are connected to the

membrane proteins studied in this thesis.

6.1 Alzheimer’s disease Alzheimer´s disease (AD) is a neurodegenerative disorder affecting millions

of people worldwide, and is the most common cause of dementia in the el-

derly population. The majority of cases correspond to the late-onset, sporad-

ic AD that develops after the age of 65. However, there is also the relatively

rare dementia that manifests at earlier age, the early-onset familial AD

(FAD). FAD is inherited in an autosomal dominant manner144; 145. Mutations

in the genes encoding the β-amyloid precursor protein (APP) and presenilins

(PS1 or PS2) are related to FAD146. Clinical symptoms of AD include loss of

memory, declining cognitive function, personality change and decreasing

physical functions147. Moreover, extracellular senile plaques and intracellular

neurofibrillary tangles defines the neuropathology of AD. The plaques con-

sist mostly of the small amyloid β-peptide (Aβ) peptide fragments derived

from APP148. APP is sequentially cleaved by β- and γ-secretase generating

Aβ149; 150. The γ-secretase is a membrane bound protease that is composed by

4 membrane proteins, presenilin 1 (PS1), nicastrin, anterior pharynx-

43

defective 1 (Aph-1) and presenilin enhancer 2 (Pen-2)151; 152. The PS1 subu-

nit contains the catalytic site of γ-secretase153. Interestingly, γ-secretase is an

aspartyl protease like the SPP, although they have different membrane orien-

tation. Furthermore, both SPP and PS1 belong to the family of i-CLiPs,

which also includes the zinc metalloprotease site-2 protease family and the

rhomboid family of serine proteases154. In this thesis we focus on studying

the FAD-linked substitution in PS1146.

6.2 Interstitial lung disease Interstitial lung disease (ILD) is a group of lung disorders that affect the

interstitium in the lungs, which is the tissue around the air sacs. As a result

the tissue becomes stiff and scarred, and the air sacs cannot expand as much

impeding the uptake of oxygen. The mammalian lung is composed of mil-

lions of alveoli, which provide an extensive surface area allowing efficient

gas exchange between epithelial cells and the capillaries in the alveolus155.

The alveoli are coated with a thin film of pulmonary surfactant, a lipid-

protein complex that reduces the surface tension at the air-liquid interface,

thereby reducing the work of breathing. Deficiency in the surfactant causes

severe respiratory disorders, e.g. ILD. The protein moiety of the surfactant is

composed of four proteins, which are divided into two groups, the hydro-

philic surfactant proteins SP-A, and SP-D, and the hydrophobic, SP-B and

SP-C156. Mutations in the SP-C gene are linked to familial and sporadic ILD,

where some of them cause misfolding and aggregates157; 158; 159; 160. SP-C is

expressed exclusively by alveolar type II cells as a 197 amino-acid precursor

protein (proSP-C) that is processed to a 34 amino-acid residue mature mem-

brane protein161. The C-terminal of proSP-C contains a linker region and a

BRICHOS domain, which is localized to the ER lumen162; 163. The TM re-

gion is Val rich, a feature that can cause folding problems, due to the low

propensity of Val in forming α-helices164; 165. This was confirmed by an SP-

C analogue, where all the Val were replaced with Leu, which resulted into a

44

more stable helix without aggregation problems166. The BRICHOS domain

has been suggested to function as a chaperone in aiding the Val rich TM

segment to form an α-helix for membrane insertion, thus preventing aggre-

gation167; 168.

6.3 Hepatocellular carcinoma The hepatitis B virus (HBV) targets the liver and replicates in hepatocytes.

The infection can develop to progressive chronic liver disease (CLD), seen

as hepatitis, fibrosis, cirrhosis, and finally heptacellular carcinoma (HCC).

HCC is the most common cancer worldwide169. The virus encodes hepatitis

B x antigen (HBxAg) that is a trans-activating protein. HBxAg seems to

alter the expression of cellular host genes that promote growth, survival and

tumorigenesis in the liver170. One of these is the up-regulated gene clone 7

(URG7), which is involved in blocking caspases via the TNFα pathway, thus

inhibiting apoptosis171; 172. Interestingly, the URG7 protein shows a promi-

nent similarity to the multidrug-resistance protein 6 (MRP6). Of the 99 ami-

no acids of URG7, the N-terminal 74 residues are identical to the N-terminal

of MRP6171. Although, the exact role of MRP6 is unknown it has been clas-

sified as a multidrug-resistance associated protein because it is highly ho-

mologous to MRP1173. Remarkably, mutations in the gene of MRP are asso-

ciated to the pseudoxanthoma elastaticum (PXE) disorder. PXE is character-

ized by progressive calcification of elastic structures in the skin, eyes and

cardiovascular system174.

45

7. Methodology

7.1 The model protein: Leader peptidase Leader peptidase (Lep) is a well-characterized protease that is located in the

inner membrane of E. coli, where it cleaves signal sequences from secretory

proteins. It has two hydrophobic domains, H1 and H2 that span the mem-

brane, and the two soluble domains, the small N-terminal (P1) and large C-

terminal (P2)175. The P2 domain faces the periplasm and contains the catalyt-

ic site and a natural glycosylation site80; 176. Lep inserts co-translationally

into the ER membrane and becomes glycosylated in the presence of micro-

somes, adopting its native conformation136 (Fig. 9). The H1 segment is more

hydrophobic than H2 and can insert into the membrane independently of H2.

In contrast, H2 needs the assistance of H1 for proper membrane integra-

tion35.

Fig. 9. Topology of the E. coli model protein Lep. Lep has two TM segments and

the N- and C-terminal faces the periplasm. The large P2 domain contains the catalyt-

ic site.

46

7.1.1 LepH2 and LepH3 Much of the work for this thesis was done on two modified versions of Lep,

LepH2 and LepH3 (Fig 10). In LepH2 the test segment replaces the H2, and

two glycosylation sites were introduced in the N-terminal and P2 domains177.

In LepH3 the test segment was introduced in the P2 domain, which is

flanked by two engineered glycosylation sites105. The test segment can be

positioned in two orientations depending on which Lep version is used.

LepH2 displays an NinCout orientation, while LepH3 inserts in an NoutCin

orientation in the ER membrane (Fig. 10). Upon expression of LepH2 and

LepH3 the glycosylation patterns between singly and doubly glycosylated

proteins can provide information about the insertion efficiency of the test

segment.

Fig. 10. Lep model protein variants. The test TM segment is in red (TMD) and have

two different orientation depending on which Lep variant is used. Left panel. LepH2

test segment inserts in an NinCout orientation, where both glycosylation sites (G1 and

G2) are modified. Right panel. LepH3 test segment inserts in an NoutCin topology

and only G2 is modified.

47

7.2 Determining the propensity to integrate into the ER

membrane The glycosylated protein species can be separated by SDS-PAGE and the

intensity of the bands in the gel are quantified. This is a direct measurement

of the inserted status of the test segment, which can be used to calculate the

apparent change in Gibbs free energy, ΔGapp. This can be seen as a simpli-

fied model where the translocon-mediated insertion process is considered

purely as an equilibrium process105; 106. The extent of inserted (!!) and non-

inserted (!!) test segment can be written as a probability (!) of insertion:

! =!!

!! + !!

and as an apparent equilibrium constant (!!""):

!!"" =!!!!

that can be placed in the equation of Gibbs free energy(ΔG!"#):

Δ!!"# = −!"   ln!!""

where ! is the gas constant and the ! absolute temperature.

7.3 Determining the topology of membrane proteins It is evident that membrane proteins are difficult to work with in respect to

the fact that they are buried inside the membrane. In order to crystalize them

they need to be isolated from the lipid bilayer, however, this is not an easy

task because they tend to aggregate. Moreover, by using detergents or artifi-

48

cial membranes to solubilize them, may affect their behavior and even dis-

turb their structure. Therefore they represent only 1% of the solved protein

structures178, due to the difficulties of overexpressing, purifying and crystal-

ize them, as discussed above. Mammalian membrane protein structures are

even more difficult to obtain because of their complexity. This is why struc-

tural biologists commonly work on bacterial homologues instead14. Howev-

er, solved structures need to be validated experimentally. There are a num-

bers of topology determinant tools available that can provide structural in-

formation experimentally. In addition, it is suitable to test membrane pro-

teins in silico, using computational tools as a first approach when only the

sequence is known of a membrane protein.

7.3.1 Topology prediction Despite the fact that membrane proteins are biologically important, very few

have had their topology determined experimentally. Topology prediction

algorithms may be helpful in providing a first approximation. They rely on

two major topological features. Firstly the hydrophobic sequence stretches,

and secondly the positive charge biases towards the intracellular side of the

membrane e.g. the ‘positive inside rule’133; 179. These algorithms provide

information about how many TM segments a protein has, including the TM

domain boundaries, and the localization of the loop domains180. However,

the increase of the crystal structures of membrane proteins has contributed

developing better prediction programs. Additionally, the number of experi-

ments dealing with membrane protein topology has increased the accuracy

of predicting topologies, which has reached the 70-80% level181. Still, the

available prediction algorithms are not completely reliable and can predict

wrong topology, much remains to be done to improve the output178.

49

7.3.2 Topology mapping Although there are a number of prediction programs for membrane protein

topology, it is necessary to verify the topology experimentally107; 182; 183; 184;

185. There are several techniques available that can provide membrane pro-

tein structural knowledge. This often involves a reporter that is fused to the

protein and reveals at which side of the membrane the fusion site resides.

However, cautions need to be taken as fusing a reporter can affect the fold-

ing and additionally the targeting if it is introduced N-terminally186. Another

type of technique is when identified target sites are introduced in the poly-

peptide, e.g. N-linked glycosylation sites, Cys residues, antibody epitopes

and proteolytic sites. Again, these sites are used to determine their accessi-

bility at one side of the membrane187. An additional approach is proteolytic

cleavage of all the loops that reside outside the membrane.

7.3.2.1 Glycosylation assay In eukaryotes the N-linked glycosylation occurs in the ER lumen by the ac-

tion of the OST complex, mentioned previously. Carbohydrates are covalent-

ly attached to the asparagine residue present in the consensus sequence Asn-

X-Thr/Ser, where X can be any amino acid except Pro188; 189; 190. The glyco-

sylation sites are introduced by site-directed mutagenesis at specific posi-

tions in the protein gene. Addition of an oligosaccharide to the target site

results in an increase of the molecular mass by approximately 2.5 kDa,

which can be detected by SDS-PAGE191. For efficient glycosylation the ac-

ceptor site should not be too close to the membrane90. The distance between

the active site of OST and the ER membrane was determined192. Additional

studies led to the minimal glycosylation distance (MGD), which is the dis-

tance required for half-maximal glycosylation, that is approximately 14 resi-

dues upstream or 10 residues downstream of the TM domain124; 193.

50

7.3.2.2 Protease protection assay The widely used Proteinase K is a non-specific protease194. It cleaves ex-

posed hydrophilic loops, while the membrane embedded segments are pro-

tected. In addition since proteolytic enzymes cannot cross the membrane the

inside loops are also protected against cleavage. The protected fragments can

be detected by SDS-PAGE, thus revealing or confirming topologies of

membrane proteins. This technique has an advantage of the fact that the

studied protein does not need any kind of modification. However, the limita-

tion of inaccessibility of the loops because of steric hindrance can give nega-

tive results187.

7.3.2.3 Glycosylatable GFP assay The Green fluorescent protein (GFP) originally comes from jellyfish Ae-

quorea victoria195. This protein is widely used as a C-terminal fused reporter.

It displays fluorescence when it is localized in the cytoplasm due to folding,

in contrast when it is translocated to the periplasm it will not196, thus provid-

ing information about the localization of the C-terminal197; 198. Extended

studies have provided the use of a glycosylatable GFP (gGFP) for eukaryotic

cells, which is a variant of GFP that contains an engineered N-linked glyco-

sylation site199. Hence, when gGFP is localized in the ER membrane and

glycosylated the fluorescence is abolished. In contrast, the gGFP displays

fluorescence in the cytoplasm.

51

8. Summary of papers

Paper I In this paper we studied PS1 that is part of the γ-secretase, an intramembrane

aspartyl protease. PS1 is involved in the processing of APP that results in Aβ

peptides, which are found as aggregates in the brain of people suffering from

Alzheimer’s disease. In addition, mutations have been found in the PS1

gene, which are connected to the more severe form of AD that appears at

earlier age, i.e. early-onset AD. It is of interest to elucidate the effect of the

FAD-linked mutations in terms of function and structure of PS1. With the N-

linked glycosylation assay we are able to address questions concerning

membrane integration. For this reason we chose a substitution located in the

TM region that can be detectable by this method. The insertion efficiency

differences between the wild type (WT) and the TM domain containing a

substitution were measured. We found that very well inserted TM segments

did not show any differences in insertion (TM1-5, TM8-9). Interestingly,

TM6, H7 and TM7, which are the hydrophobic domains that constitute the

catalytic site, were more susceptible for substitutions. As expected, hydro-

phobic to polar residue substitution resulted in membrane integration de-

crease, the opposite effect was obtained when polar residues were substituted

to hydrophobic ones. Moreover, the substitution that showed effect were

expressed and tested in vivo for functionality. The ratio between the Aβ42

and Aβ40 was measured. We observed that changes in the membrane inte-

gration did not correlate well with the increase in the Aβ42/ Aβ40 ratio but

did show conformational changes in the active site. The FAD-linked muta-

52

tions may affect the PS1 molecule in various ways and there is not only one

mechanism that is behind the AD pathogenesis.

Paper II Here we study the novel protein URG7, which is one of the proteins that is

up regulated during the development of liver cancer. It has been suggested to

be involved in blocking caspases, which leads to the inhibition of apoptosis.

URG7 has a natural glycosylation site on the N-terminal. In order to assess

the topology additional acceptor sites were introduced. We propose that it

anchors the membrane in an NoutCin manner, consistent with the orientation

of MRP6. The truncated MRP6 (TM1-TM2) with known topology was used

as a control for ER targeting. URG7 has the N-terminal 74 residues identical

to the N-terminal of MRP6. Moreover, URG7 was expressed in HepG2 cells

and we found that it is glycosylated and localized to the ER in agree with the

in vitro studies.

Paper III In this paper we study the SP-C, which is part of the lung surfactant that is

involved in reducing the surface tension at the air-liquid interface in the

lung. SP-C is associated with lung diseases; mutations found in the gene of

SP-C can result in amyloid deposits in the lung. The SP-C is synthesized as a

197 residues proprotein (proSPC-C) and is proteolytically processed to a

hydrophobic 35 amino-acid peptide. The TM domain of this protein is main-

ly composed of Val residues, which has a high propensity to form β-strands.

The C-terminal contains a chaperon-like domain, the BRICHOS domain that

is thought to aid the TM segment to fold correctly and insert into the mem-

brane. In contrast, if the TM segment is not properly folded it will instead

convert into β-sheet aggregates and amyoid-like fibrils. A synthetic SP-C

analogue, where all the Val residues were substituted to Leu, resulted in a

53

more stable TM domain. Here we study the folding of SP-C (poly-Val) and

its analogue (poly-Leu) by introducing N-linked glycosylation sites in the C-

terminal of the protein at different distances from the TM segment. We

found that poly-Val gets more efficiently glycosylated at closer distances

from the membrane than poly-Leu. This suggests that poly-Val is in a more

extended conformation, while poly-Leu adopts a more compact, folded con-

formation. We also study the interaction of membranes with the WT

BRICHOS and an ILD-associated variant (L188Q) in order to better under-

stand how BRICHOS execute its role as a chaperon. We could see that WT

BRICHOS can insert into the membrane, probably into the head group re-

gion, while no insertion was observed for L188Q. This suggests that mem-

brane insertion of the BRICHOS domain is important for the binding to the

misfolded proSP-C. Moreover, the insertion may change the property of the

membrane locally, preventing β-sheet formation.

Paper IV In this paper we developed the use of gGFP in mammalian cells as a mem-

brane protein topology reporter. The gGFP is C-terminally fused to prefera-

ble single-spanning membrane proteins. The engineered gGFP contains two

glycosylation sites that upon modification in the ER loose the fluorescence.

On the other hand, if located in the cytosol the proteins becomes fluorescent

and non-glycosylated. By measuring the fluorescence and assessing the gly-

cosylated state of a gGFP fusion protein, the localization of the C-terminal

part of the protein can be determined. Here we confirm the topology of the

four clinically important membrane proteins analysed in paper II and III. The

URG7 and MRP6 were fluorescent and non-glycosylated respectively non-

fluorescent and glycosylated, displaying an NoutCin and an NinCout topology.

In addition both SP-C and its analogue were glycosylated but did not exhibit

fluorescence, having the C-terminal in the lumen. gGFP can easily be used

to assess topologies of membrane proteins in mammalian cells.

54

9. Conclusions and perspectives

Membrane protein is the main focus in this thesis, especially those that are

connected to diseases are particularly interesting for us. It is common to hear

researchers say that membrane proteins are difficult to work with, the rea-

sons are discussed above, and many avoid working with them for that. How-

ever, they are extremely important and the interest for them arises further

when realizing their involvement in diseases. They become malfunctioned

by for example acquiring a mutation in their gene that lead to loss of func-

tion. In addition the membrane protein can in worse case aggregate and in-

fect other proteins, causing them to aggregate as well. This reaction can lead

to cell death, and later on to tissue lost that can become life threatening. A

critical step toward elucidation of their roles in the complex processes is the

understanding of their structure and function.

In the first paper we study PS1, which is involved in AD. This disorder is

spread all over the world and is one of the most financially costly diseases.

The well-studied PS1 has been accepted to play a key role in the disease,

mutations has been found in the gene of PS1 that cause worse cases of AD.

In this case the topology of PS1 is known, so we focus in studying what

effect the mutations have in terms of structure and function. We learnt that in

many cases they do not correlate to each other. However, there are limita-

tions in the method used, the FAD-linked mutations where studied in indi-

vidual TM segments, lacking the rest of the protein. We can suggest what

effect the specific substitution will have on the specific TM segments where

it sits. However, the TM segment containing the substitution is in reality

surrounded by the other PS1 TM segments, which may shield and lowered

55

the effect. In addition, they might cause rearrangements in the whole protein

that we cannot detect, causing it to loose its function. However, these chang-

es can be very small to perceive, even though the effect is immense.

In the case of URG7 protein, both the topology and function is unknown, but

it has been suggested to be involved in the progress of liver cancer. Liver

cancer is one of the most frequent cancer globally that cause death. We sug-

gest that URG7 is targeted to the ER, and it spans the membrane once with

the N-terminal in the lumen. In this case we work with the full-length protein

that has a natural glycosylation site in the N-terminal, which is modified by

the OST complex. In order to verify these results we introduce further sites

that confirm the NoutCin topology of the protein. Moreover, the protein is

expressed in vivo to find its localization, which is in the ER.

The importance of synthesizing, inserting, folding and sorting the membrane

protein correctly is crucial for its function. Misfolding and aggregation can

occur during the process of maturation that chaperons and protein degrada-

tion system present in cells can take care of. However, this can be fatal if the

system is not completely working and is overloaded with protein aggregates.

An example of membrane protein that has the ability to aggregate is the SP-

C, which is involved in lung diseases. This protein is essential for our lungs

to work properly. Mutations found in the gene of this membrane protein can

result in aggregation and respiratory failure. Interestingly, this protein con-

tains a chaperon-like domain, the BRICHOS domain that might be required

for correct folding of the TM segment. The SP-C TM domain is special in

the sense of that it is composed of mostly Val and Ile, residues not common-

ly found in α-helices. The Val rich TM domain might need assistance from

the BRICHOS domain to fold correctly and insert into the membrane. We

studied the biogenesis of proSP-C and its analogue, which has all the Val

replaced by Leu. As expected, we could show that the poly-Leu is better

56

suited to fold than poly-Val. We could also show that the BRICHOS domain

interacts with the lipid bilayer suggesting its involvement as a chaperone

aiding the TM segment to fold.

In the fourth paper, we use gGFP as a reporter protein to confirm the topolo-

gies of URG7, MRP6102, proSP-C (poly-Val) and proSP-C (poly-Leu) in

vivo. Since the gGFP is fused C-terminally it can only report the localization

of the C-terminal, which is highly useful for single-spanning membrane pro-

teins. However, for multi-spanning it is not as suitable, as for PS1 that has

nine TM segments. Knowing the C-terminal location of a multi-spanning

membrane protein is not enough to know its whole topology. One way to

overcome this problem is to truncate the protein from the C-terminal, but it

is always preferentially to use the full-length protein instead of truncated

ones.

57

Acknowledgements

I would like to thank all the people that have made this thesis possible and that have been present during these 5 years of PhD studies. Even though it has been like a roller coaster, it is the great moments that will be memorized. First of all I have to thank my supervisor IngMarie Nilsson. Tack så mycket för all hjälp som jag har fått utav dig. Det har varit prisvärt att få ta del av din erfarenhet. Tack för alla mysiga och goda luncher J. Jag är även tack-sam för chansen att ha fått jobba utomlands, vilket har varit väldigt lärorikt. Min andra handledare Gunnar von Heijne, för att du har gjort det möjligt för mig att doktorera i DBB och för att få mig känna som en del av din grupp. Tack Stefan för alla dina inputs, för att du finns alltid där för oss doktoran-der. Tack Elzbieta, Joe, Peter mina entusiastiska utvärderade! All co-authors for sharing their knowledge, Johanna Wanngren and Helena Karlström, tack för alla diskussionerna angående PS1. Magnus Monné för att ha fått chansen att jobba med det spännande URG7. Janne, tack för alla förklaringar vad gäller SP-C. Jenny, alltid positiv, ditt intresse för science är verkligen inspirerande och smittar av sig J. Joy I’m really thankful for the time in South Korea, for letting me work in your lab, and also for the time spent outside the lab. And as I told you, you have a wonderful group! Hunsang, thank you for being such a nice lab mate and also a friend! I’m so happy that our work could be published while we had lots of fun in and out-side the lab J. The people from SouthKorea: Jake, first of all thank you for introducing me to bachata J. It was great to have you here in Stockholm, and thanks for all the help in Korea, for showing me the fun side of Seoul. Sungjun, my sweet-ie! Thank you for always being so helpful and always with a smile, you are amazing! Sungminaaa and Hayoung, my best teachers in Korea kkkk. My

58

girls: Hani, Chewon, Ramla and Kyungyeun, thank you for showing me Seoul!! Johannes, you know whatever happened in Korea stays in KoreaJ. Many thanks to the people in the lab and office mates from the past and pre-sent. Carolina, Ola, Nir! Åsa, tack för att du läste min avhandling. Karin tack för allt du har lärt mig, och alla samtal. Jag kommer aldrig glömma vår resa till Japan med KatrinJ. Jätte kul att vi blir klara samtidigt. Lycka till med allt! Nina, vi har undervisat, rest och spenderat många luncher tillsam-mans det enda som fattades var att jobba ihop. Men vem vet, kanske i fram-tiden J tack för alla goda minnen, speciellt roadtripen! Florian thank you for always sharing papers and your knowledge! My dear Salome I miss you so much but even though you’re far away I can still talk to you about every-thing, thank you for always listening, te quiero mucho <3! Susanna, ditt sätt att prata science har alltid varit inspirerande vilket man har saknat! Tack för alla dina tips. The GvH people from the past and present: Thank you for the all the good times!! Luigi, Grant, Renuka!! Rickard, tack för alla roliga stunder vi har haft i och utanför DBB. Och för våra långa konversationer som kan handla om vad som helst J. Bill, muchas gracias för din eviga uppmuntran och för den sköna musiken som hördes ända in i vårt labb. Carmen gracias por tu compañia que mucho se aprecia! Pili!! Gracias chica por el apoyo en espe-cial este año de estrés.!! Thank you so much Nurzian for all this time!! First for introducing me to salsa, not the one that I was used to, and also for intro-ducing me to all your nice Singaporean friends: BJ, Kyle, Mariam, Sharon, will never forget the trip to Beijing and the Great Wall, it was amazing! Jimmie!! The pole-dancing queen!! Lol To my students I wish you all the best! Manuel, Laura, Lu. Nassim lycka till med jobbsökandet!. Aurora, mi niña!! Te tome un monton de cariño por ser tan especial J. And to people that worked in our lab for shorter period: Carlos fue demasiado entretenido tenerte con nosotros! Sweet Flavia you are soon leaving! Good luck! Maikel, my crazy friend J I will visit you soon! The GvH community: Jan-Willem group that is always so nice! Zhe, Grietje. Anna, kommer aldrig glömma alla fina minnen från och utanför DBB. Häl-sosamma minnen som vårruset men även roliga som spybarJ. Thomas you always look so happy, keep up with the good energy! The cool group of Dan

59

D!! Rageia. Claudio, it’s always fun to talk to you and also danceJ. Kiavash, blir glad av att se hur mycket du har utvecklats från Biokemi 1 till nu!! Stephen, alltid lugn och snäll J. Mikaela tack för du delar med dig av din erfarenhet. Kimmo always so kind!! Söta Hena! Alltid lugn och ordent-ligJ. David Drew group: Mathieu, Emmanuel, Abdul, Povilas. Neighbours in DBB!!! Rob Daniels, thank you for letting me work in your lab and for nice discussions!! Johan tack för tålamodet att visa mig vad allt ligger i ert labb! Diogo, Hao, Dan you guys are always so nice!! Candan my friend! Thank you for your support and for our fika times, the Turkish coffee that I loveJ! Catarina you are really missed! Your kindness and energy is irreplaceable! Erika tack för alla dina kloka ord när man behövde de som mest! The cute couple Pedro and Beata!! Emelie, din glädje i DBBs korri-dorer är verkligen uppfriskande, du får mig alltid att le J. Martin Ott and group, for always being so kind! Lisa tack för alla samtal!! Sylvia och Patrik, kommer aldrig glömma SpetsesJ Nikolas, for the nice talks in the corridor! Mina tjejer från stora tentan pluggandet och undervisning! Narges, Gabriella, Minttu! Det är alltid så härligt att umgås med er, allt ni delar med er men även för att ha lyssnat på mina issues. Det har alltid varit skönt att kunna fly till er om jag behöver det J. Många kramar, ni är bäst <3!! Till alla på sekretariatet, tack för att ni har varit så hjälpsamma! Ann, Lotta, Maria, Alex, Anita och Haidi! Får inte heller glömma, Håkan, Peter och Torbjörn för att alltid ställa upp om det är något man behöver!! Människor utanför DBB som nog aldrig har riktigt förstått vad jag har jobbat medJ! Kalle för att du trodde på mig och gav mig stödet jag behövde för att påbörja det här, fick lära mig otroligt mycket av dig. Önskar dig all lycka! Fatma, una de las personas mas linda que he conocido! Gracias por tus consejos cuando mas lo necesitaba, BESO! Rojda, min kloka vän, vet inte vad jag hade gjort utan dig! Älskar våra samtal!! Love YOU <3! Mina barn-domsvänner Evelyn och Pabla, ni har alltid funnits där J. Mina fina brudar Claudia och Hevi! puss på er! Mi amiga de siempre y prima Claudia TQM! Mis amigas del baile, que siempre me alegran y me hacen pensar en otras cosas fuera del trabajo J Yasmin, mi amore siempre tan alegre y linda! Te quiero mucho! Sihomara, la India fue increible, gracias por haber compar-tido ese viaje conmigo. Veronica! Shirley! Lissan! Pamela! las quiero mucho

60

y aprecio mucho las conversaciones con uds! Mina bachata killar, Hebbe, Felipe, Johan, Oskar y Luis - gracias por haber estado ahi cuando lo ne-cesitaba y mi primito que mucho quiero Miguel! Sammy för att du glädjer min vardag! Mis pastelesJ!! Jocelyn y Kenia gracias por todos los buenos momentos, uds saben ;)! La familia Lara y Vasquez aqui en Suecia y tambien en Chile!! Tios, tias y primitos e hijos de los primos! No puedo nombrarlos a todos porque son muchos J!! Pero los quiero a todos!! Mi familia que lo son todo! Mis hermanitos Ivan y Marite, estoy muy orgul-lasa de uds y los amo! Muchas gracias por mis sobrinos preciosos Jamilia y Matthias J y sus papitos Sinan y Alicia. Mamita y papito, gracias por haber siempre confiado en mi! Sin uds no estaria aqui! Los amo mucho!! Mi padre celestial que me da la sabiduria y fuerza <3!

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