" Role Of In Endoplasmic Reticulum Stress Response In Sacccharoromyces cerevisiae ".

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ROLE OF Ncr1p IN ENDOPLASMIC RETICULUM STRESS

RESPONSE IN Saccharomyces cerevisiae

JOANA FILIPA MADUREIRA GAIFEM

Dissertação de Mestrado em Contaminação e Toxicologia Ambientais

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JOANA FILIPA MADUREIRA GAIFEM

ROLE OF Ncr1p IN ENDOPLASMIC RETICULUM STRESS

RESPONSE IN Saccharomyces cerevisiae

Dissertação de Candidatura ao grau de

Mestre em Contaminação e Toxicologia

Ambientais submetida ao Instituto de

Ciências Biomédicas de Abel Salazar da

Universidade do Porto.

Orientador – Doutor Vítor Costa

Categoria – Professor Associado

Afiliação

– Instituto de Biologia Molecular e

Celular; Instituto de Ciências Biomédicas de

Abel Salazar

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Acknowledgements

O trabalho apresentado nesta tese contou com o contributo importante de várias pessoas, sem as quais a sua realização seria impossível ou estaria severamente condicionada. Como tal, gostaria de lhes expressar os meus agradecimentos.

Em primeiro lugar, gostaria de agradecer ao meu orientador, o Professor Doutor Vítor Costa, por ter aceitado orientar-me neste trabalho e por todos os contributos que deu para a elaboração desta tese. Todos os conselhos, ensinamentos e correcções foram essenciais para o desenvolvimento deste trabalho e para o meu crescimento profissional. Muito obrigado Professor!

Agradeço à Rita Vilaça, que co-orientou este trabalho e que contribuiu de forma fundamental para a realização do mesmo. Todas as interacções tiveram o seu papel na minha aprendizagem, na criação de dinâmica de trabalho e na evolução do projecto. Por todos estes motivos e pela disponibilidade demonstrada ao longo de todo o ano de trabalho, o meu obrigado.

Porque o companheirismo e bom ambiente contribuem de forma notória para a elaboração de um bom trabalho, quero expressar a minha gratidão a todos os elementos do laboratório com quem pude conviver ao longo deste ano de trabalho. Ao Daniel, agradeço por toda a disponibilidade em ajudar em diversos aspectos do meu trabalho, assim como pelos conselhos dados na elaboração desta tese, e, não menos importante, agradeço pela amizade espontânea e pelas conversas diárias, no laboratório ou fora dele. Quero agradecer à Vanda, não só pelo convívio, mas também pelos contributos que deu ao meu trabalho. Agradeço à Catarina Santos, que se revelou uma boa amiga, sempre com uma palavra certa no momento certo, para além dos momentos de descontracção. À Sílvia, agradeço toda a alegria que transmite no laboratório, bem como a disponibilidade para ajudar no necessário. Quero agradecer à Catarina Pacheco e à Maria João pelas conversas diárias que proporcionaram momentos agradáveis e descontraídos. À Sara Silva e ao João Ferreira, que partilharam comigo a experiência de um ano de trabalho para a elaboração de uma tese de mestrado, o meu obrigado. Agradeço ao Prof. Dr. Pedro Moradas-Ferreira pelo interesse demonstrado no trabalho. Não posso deixar de agradecer a todos os elementos dos grupos de Redox Cell Signalling, Cellular and Applied Microbiology e Bioengineering and Synthetic Microbiology

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pelo companheirismo e pelo excelente ambiente de trabalho. À Ângela, Filipe, Gabriel, Ivan, Kalina, Marta, Miguel, Nuno, Prof. Dr.ª Paula Tamagnini, Prof. Dr.ª Arlete Santos, Pedro, Rodrigo, Rita Mota, Sara Pereira, Tiago, Vítor e Zille, muito obrigado. Deixo também os meus agradecimentos à Liliana Correia e à Helena Pinho, pela simpatia e pela disponibilidade em ajudar no que fosse necessário.

A todos os meus amigos, o meu muito obrigado! Todos contribuíram para a realização deste trabalho, pelo apoio e confiança que sempre me transmitiram. Um agradecimento especial à Ana Luísa e ao Tiago Miguel, que mesmo à distância sempre me apoiaram e de quem me orgulho de ser amiga. Aos meus colegas de mestrado, agradeço a amizade e os bons momentos partilhados neste ciclo novo para todos nós, e em particular, à Alexandra, com quem construí uma bela amizade e com quem partilhei momentos de grande alegria e companheirismo.

Como não pode deixar de ser, quero agradecer a toda a minha família por todo o apoio e confiança que me deram, e em particular aos meus avós António e Ana. Graças ao seu apoio, não só durante este ano, mas ao longo de toda a minha vida, e a todos os ensinamentos que me transmitiram, pude crescer como pessoa e chegar mais longe. Teria sido muito mais difícil sem eles. Muito obrigado por tudo!

Por fim, agradeço às pessoas que convivem comigo diariamente e que acompanharam mais de perto todos os meus passos. Aos meus pais, agradeço-lhes do fundo do coração todo o carinho e confiança que sempre me transmitiram. Não há palavras suficientes para descrever a gratidão que sinto por tudo o que fizeram por mim. Em todos os momentos, não só este ano, mas ao longo de toda a minha vida, estiveram presentes para me dar a mão sempre que precisei, para me mostrar o que está certo e o que está errado, para me tornarem numa pessoa digna de chegar cada vez mais longe. Devo-vos tudo o que sou. Ao meu irmão Bruno, dedico-lhe esta tese, pois desde o dia que nasceu que é a minha maior inspiração. Todos os momentos de brincadeira, de amizade e de apoio foram preponderantes para me dar a força necessária para os momentos mais importantes. É o melhor irmão do mundo e tenho muito orgulho na pessoa maravilhosa que é. Por último, quero agradecer ao Filipe, por todas as razões. Pela confiança, pela amizade, por estar sempre ao meu lado, em momentos bons e menos bons. É uma honra poder caminhar ao seu lado e com o seu apoio sei que chegarei mais longe. Muito obrigado por tudo!

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Figure Index

Figure I.1. Lipid trafficking defects in NPC disease.. ...18

Figure I.2. Topology of NPC1 ...19

Figure I.3. The Ire1p cascade of the UPR pathway ...27

Figure I.4. The High Osmolarity Glycerol (HOG) pathway.. ...31

Figure IV.1. The ncr1Δ cells are resistant to tunicamycin.. ...51

Figure IV.2. Effect of tunicamycin on cell growth. ...52

Figure IV.3. Analysis of tunicamycin resistance. ...53

Figure IV.4. Analysis of ROS levels. ...54

Figure IV.5. Effect of tunicamycin on glutathione levels. ...55

Figure IV.6. Structure of UPRE-lacZ gene reporter. ...56

Figure IV.7. Hac1p activation by ER stress ...57

Figure IV.8. HAC1 deletion increases the sensitivity of ncr1Δ cells to tunicamycin ...58

Figure IV.9. Analysis of CPY* stability ...59

Figure IV.10. Quantification of CPY* decay. ...59

Figure IV.11. Hog1p phosphorylation is decreased in ncr1Δ cells ...60

Table Index

Table III.1. Yeast strains used in this work. ...39

Table III.2. Primers used in this work. ...41

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Abbreviations

ATF – Activating Transcription Factors ATF6 – Activating Transcription Factor 6 ATP – Adenosine Triphosphate

BSA – Bovine Serum Albumin bZIP – Basic Leucine Zipper Domain cAMP – Cyclic Adenosine Monophosphate CDP – Cytidine Diphosphate

CFU –Colony Forming Units CH - Cycloheximide

CNS – Central Nervous System CPY* – Carboxypeptidase Y

CREB – cAMP Response Element Binding DMSO – Dimethylsulfoxide

DNA – Deoxyribonucleic Acid dNTP – Deoxyribonucleotides

DTNB – 5,5'-Dithiobis-(2-Nitrobenzoic Acid) DTT – Dithiothreitol

EDEM – ER Degradation-Enhancing α-Mannosidase-like Protein EDTA – Ethylenediaminetetraacetic Acid

ER – Endoplasmic Reticulum

ERAD – Endoplasmic Reticulum-Associated Degradation GFP – Green Fluorescent Protein

GLS – Golgi Localization Sequences GSH – Glutathione (reduced form) GSSH – Glutathione (oxidized form) GST – Glutathione S-Transferases HOG – High Osmolarity Glycerol

H2DCF-DA – 2’-7’-Dichlorodihydrofluorescein diacetate

IRE1 – Inositol-Requiring Protein 1 LDL – Low-Density Lipoprotein

MAPK – Mitogen-Activated Protein Kinase MOPS – 4-Morpholinepropanesulfonic Acid mRNA – Messenger RNA

NADPH – Nicotinamide Adenine Dinucleotide Phosphate NPC – Niemann-Pick type C

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OD – Optical Density

ONPG – o-nitrophenylgalactopyranosyde PAGE – Polyacrylamide Gel Electrophoresis PBS – Phosphate Buffered Saline

PCR – Polymerase Chain Reaction PDI – Protein Disulfide Isomerases

PERK – Protein kinase RNA-like ER kinase PMSF – Phenylmethylsulfonyl Fluoride RNA – Ribonucleic Acid

rpm – Revolutions Per Minute ROS – Reactive Oxygen Species

SAPK – Stress-Activated Protein Kinase SC – Synthetic Complete

SD – Standard Deviation SDS – Sodium Dodecyl Sulfate SSD – Sterol Sensing Domain

TEMED – N,N,N,N-Tetramethylethylenediamine TOR – Target of Rapamycin

TPBS – Tween Phosphate Buffered Saline tRNA – Transfer RNA

TBS – Tris Buffered Saline

TTBS – Tris-Tween Buffered Saline TUN - Tunicamycin

UPR – Unfolded Protein Response

UPRE – Unfolded Protein Response Element UPS – Ubiquitin-Proteasome System

UTR – Untranslated Region wt – wild-type

XBP1 – X-box Binding Protein-1 YPD – Yeast extract Peptone Glycerol

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Abstract

Niemann-Pick Type C (NPC) is an autossomal recessive lipid storage disease characterized by abnormal cholesterol trafficking and intracellular accumulation in late endosomes and lysosomes. NPC disease is related to a progressive neurodegenerative phenotype and is caused by loss-of-function point mutations in either NPC1 or NPC2. Both proteins seem to regulate intracellular lipid transport through lysosomes and endosomes. Several lipid disorders display evidences of endoplasmic reticulum (ER) stress. Cell adaptation to ER stress is mediated by the unfolded protein response (UPR). This signal transduction pathway detects unfolded proteins in the lumen of ER and reduces stress by increasing the folding capacity of ER or triggers apoptosis of irreversibly damaged cells.

In Saccharomyces cerevisiae, the vacuolar proteins Ncr1p and Npc2p are orthologues of human NPC1 and NPC2, respectively. Yeast Ncr1p and Npc2p are involved in ergosterol trafficking and can functionally complement the loss of function of human NPC1 and NPC2, being able to suppress lipid trafficking defects associated with NPC1 and NPC2 mutations. Therefore, studies using yeast as an eukaryotic model may be useful to uncover the function of these proteins and to characterize molecular mechanisms associated with NPC disease.

In this study, S. cerevisiae ncr1Δ mutant cells were used as a model system to study the role of Ncr1p in ER stress response. Parental and ncr1Δ cells were treated with tunicamycin, a drug that inhibits protein glycosylation and consequently activates UPR. The results showed an increased resistance of ncr1Δ cells to tunicamycin and that Ncr1p deficiency seems to have protective effects to yeast cells from tunicamycin-induced growth arrest. The analysis of oxidative stress markers showed that tunicamycin specifically decreased glutathione levels in parental cells, but not in ncr1Δ mutants. However, ncr1Δ cells exhibited higher levels of reactive oxygen species. Notably, the induction of a reporter gene controlled by Hac1p, the transcription factor involved in the UPR, was suppressed in ncr1Δ cells exposed to tunicamycin. This effect is specific for this drug since UPR was induced in both parental and ncr1Δ cells treated with dithiothreitol (DTT), a compound that impairs disulfide bond formation. The hac1Δncr1Δ double mutant displayed a higher sensitivity to tunicamycin when compared to ncr1Δ mutant, but was more resistant than hac1Δ cells, suggesting that, for tunicamycin exposure, lack of NCR1 has protective effects by a Hac1p-independent mechanism. The analysis of endoplasmic reticulum-associated degradation (ERAD) showed that after 1 h of tunicamycin exposure this system is induced in ncr1Δ mutant cells but not in parental cells, in which tunicamycin inhibits ERAD system by saturation of its capacity. The study of the High Osmolarity

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Glycerol (HOG) pathway showed that Hog1p and phospho-Hog1p levels increased with tunicamycin exposure in parental cells, but not in ncr1Δ mutant cells, indicating that the resistance of ncr1Δ cells to tunicamycin is Hog1p-independent. These data suggest that Ncr1p deficiency increases ER stress resistance induced by tunicamycin exposure via an uncharacterized mechanism.

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Resumo

Niemann-Pick tipo C (NPC) é uma doença lipídica autossómica recessiva, caracterizada por disfunções no tráfego de colesterol e a sua acumulação a nível intracelular nos endossomas e lisossomas. A degeneração neurológica está associada a esta doença, sendo provocada por mutações pontuais nos genes NPC1 ou NPC2. Estas proteínas aparentam regular o transporte intracelular de lípidos através dos lisossomas e endossomas. Diversas doenças relacionadas com distúrbios lipídicos apresentam evidências de stress do retículo endoplasmático. A adaptação celular ao stress do retículo é mediada pela resposta à acumulação de proteínas mal conformacionadas, designada por “unfolded protein response” (UPR). Esta via de transdução do sinal detecta proteínas com conformações incorrectas no lúmen do retículo e, através do aumento da capacidade de conformação do retículo, reduz o stress ou, caso os danos celulares sejam irreversíveis, inicia o processo de apoptose.

Na levedura Saccharomyces cerevisiae, as proteínas vacuolares Ncr1p e Npc2p são ortólogas da NPC1 e NPC2 humanas, respectivamente, e estão envolvidas no transporte e tráfego de ergosterol. Ambas as proteínas Ncr1p e Npc2p podem complementar a perda de função das respectivas proteínas humanas, suprimindo as anomalias ao nível do tráfego lipídico associadas às mutações em NPC1 e NPC2. Como tal, a utilização da levedura como modelo eucariótico pode ser vantajosa para o estudo das funções dessas proteínas e para a caracterização de mecanismos moleculares relacionados com a doença de NPC.

Neste trabalho, foram usados mutantes de S. cerevisiae ncr1Δ como modelo para o estudo do papel da Ncr1p na resposta ao stress do retículo. Células parentais e do mutante ncr1Δ foram tratadas com tunicamicina, um composto que inibe a glicosilação de proteínas e consequentemente activa a UPR. Os resultados demonstraram uma maior resistência do mutante ncr1Δ à tunicamicina e a deficiência em Ncr1p diminui a inibição do crescimento induzida por este composto. A análise de marcadores de stress oxidativo mostrou uma diminuição nos níveis de glutationa induzida pela tunicamicina nas células parentais, contrariamente ao observado nos mutantes ncr1Δ. Todavia, estes mutantes apresentaram níveis de espécies reactivas de oxigénio superiores aos das células parentais. A indução de um gene repórter controlado pela proteína Hac1p, factor de transcrição associado à UPR, através da exposição com tunicamicina, foi suprimida nos mutantes ncr1Δ. Este efeito é específico para a tunicamicina uma vez que a UPR foi induzida nas células parentais e no mutante após tratamento com ditiotreitol (DTT), um composto que compromete a formação de ligações dissulfureto. O duplo mutante ncr1Δhac1Δ apresentou uma maior sensibilidade à tunicamicina comparativamente ao

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mutante ncr1Δ, mas também uma maior resistência que o mutante hac1Δ, o que sugere que a delecção do gene NCR1 surte efeitos protectores por um mecanismo independente do HAC1. Através da análise do sistema de degradação associada ao retículo endoplasmático (ERAD) verificou-se que após uma hora de exposição à tunicamicina, este sistema é induzido nos mutantes ncr1Δ, ao contrário do que sucede nas células parentais, nas quais a tunicamicina inibe o sistema de ERAD devido à saturação da sua capacidade. O estudo da via de alta osmolaridade do glicerol (HOG) demonstrou que os níveis de Hog1p e de Hog1p na forma fosforilada aumentaram com exposição à tunicamicina nas células parentais, mas não nos mutantes ncr1Δ, o que parece indicar que a resistência dos mutantes ncr1Δ é independente da proteína Hog1p. Estes dados sugerem que a deficiência na proteína Ncr1p aumenta a resistência ao stress do retículo endoplasmático induzido pela exposição à tunicamicina através de um mecanismo não identificado.

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Chapter I

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I-1. Niemann-Pick type C disease

Sphingolipid storage diseases are a group of approximately forty genetic disorders, caused by inherited defects of lysosomal hydrolytic processes or lipid transport that leads to intracellular accumulations of cholesterol and lipids in the endosomal-lysosomal system (Pacheco & Lieberman, 2008). Among this group is Niemann-Pick disease.

Niemann-Pick type C (NPC) disease, along with types A and B, belongs to the Niemann-Pick group of lipidoses (Ikonen & Holtta-Vuori, 2004). This group of diseases was first described in the late 1920’s by Albert Niemann and Ludwig Pick, as a heterogeneous group of autossomal recessive lysosomal lipid storage disorders, with or without neurological involvement, with regular features of hepatosplenomegaly and sphingomyelin storage in reticuloendothelial and parenchymal tissues. It was later demonstrated that there is a broad variability in age of onset, clinical expression and in the level of sphingomyelin storage in tissues (Crocker & Farber, 1958), which led to a classification of the disease into different groups (Crocker, 1961). Types A and B are caused by loss-of-function mutations in the acid sphingomyelinase gene (Vanier & Millat, 2003). Type C was described as having a sub acute nervous system involvement, with moderate/slower course and a mild visceral storage; however, later work led to a reclassification of type C as a cellular lipid trafficking disorder, involving more specifically endocytosed cholesterol (Pentchev et al., 1994). In NPC disease, cells fail to esterify exogenously added cholesterol. This disorder is characterized by unique abnormalities of intracellular transport of endocytosed cholesterol with accumulation of unesterified cholesterol in endosomal/lysosomal compartment and the Golgi complex (Ikonen & Holtta-Vuori, 2004; Vanier, 2010). Besides cholesterol sequestration, NPC cells can also accumulate other lipids, in particular sphingolipids (Lusa et al., 2001; Puri et al., 1999; Vanier, 1999; Zhang et al., 2001b). NPC is related to a progressive neurodegenerative phenotype and in most cases is fatal (Patterson et al., 2001).

Advances in the knowledge of the disease led to the description of two genetic complementation groups and the subsequent isolation of the two underlying genes: NPC1 and NPC2. They are represented in different proportions in the population – NPC1 is involved in 95% of the cases (Patterson et al., 2001), while NPC2 is related to rare cases (Vanier, 2010). NPC is caused by loss-of-function mutations in either NPC1 or NPC2 proteins, which mediate proper intracellular lipid transport through pathways that remain unclear (Pacheco & Lieberman, 2008).

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I-1.1. Epidemiology

The prevalence of NPC disease is difficult to assess, due to insufficient clinical awareness and difficult diagnosis. Estimates of birth prevalence for Western Europe have been predictable to be 1 per 150,000 (Patterson et al., 2001). In Australia (Meikle et al., 1999), The Netherlands (Poorthuis et al., 1999) and Portugal (Pinto et al., 2004) the prevalence is 0.71, 0.53 and 3.3 per 150,000 births, respectively.

I-1.2. Clinical description and diagnosis

The clinical presentation of NPC is extremely heterogeneous, with patients developing symptoms over a wide range of ages (Patterson et al., 2001). There is no exact correlation between disease-causing mutations and the degree of severity of the clinical phenotype (Vanier & Millat, 2003; Yamamoto et al., 2000). Similarly, the lifespan of the patients varies between a few days (Spiegel et al., 2009) until over 60 years old (Trendelenburg et al., 2006). This disease can be subdivided in four groups concerning to the age of onset: early infantile, late infantile, juvenile and adult form of the disease. However, the classic form of NPC, which encompasses approximately 70% of the cases, presents between the ages of 3 and 15 years (Patterson et al., 2001). NPC severely targets internal organs (mostly liver and spleen) and the first symptoms usually described are hepatosplenomegaly (that seems to fluctuate and decrease with time) or obstructive jaundice. The systemic involvement is usually severe, except for the perinatal period, which is well tolerated (Vanier, 2010). Nevertheless, patients eventually develop neurological and/or psychiatric symptoms, the severity of which is inversely associated with lifespan (Imrie et al., 2002; Turpin et al., 1991).

The diagnosis of NPC disease is based on the analysis of dermal fibroblasts, with two different approaches: a morphological approach, by filipin staining to detect the accumulation of free cholesterol; and a biochemical approach, to monitor defective cholesterol esterification in low density lipoprotein (LDL)-challenged cells (Vanier et al., 1991). Currently, there are no effective treatments available to patients with this disorder (Pacheco & Lieberman, 2008).

Despite the heterogeneity of the clinical symptoms of NPC disease, it is not totally observed when it comes to the biochemical level of the disease. The majority of cases present prominent accumulations of unesterified cholesterol, sphingolipids and complex gangliosides in late endosomes and lysosomes, but a subset of patients with specific mutations reveals less lipid storage (Millat et al., 2001a).

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17 The development of NPC is characterized by a liver and spleen enlargement, caused by the presence of lipid-laden macrophages. Kupffer cells in the liver and splenic macrophages display clear cytoplasmic vacuolization that results from the accumulation of cholesterol, phospholipids and glycolipids (Pacheco & Lieberman, 2008). Impairment of lipid trafficking also has severe consequences in the central nervous system (CNS), leading to neuron loss throughout the brain (Walkley & Suzuki, 2004). The presence of swollen neuronal cell bodies in many regions in the brain is also a feature of NPC and reflects lipid accumulation in late endosomes and lysosomes. It is observed also in NPC an intracellular aggregation of the microtubule-binding protein tau, which is biochemically similar to aggregates in Alzheimer’s disease (Auer et al., 1995).

I-1.3. Lipid-trafficking defects in NPC

Lipid-trafficking defects within the NPC brain reflect deficiencies in the pathway by which cholesterol and other lipids reach neurons and are sorted intracellularly (Pacheco & Lieberman, 2008). Neurons and other CNS cell types get cholesterol they need through endogenous synthesis or by uptake of lipoprotein cholesterol particles produced and released within the nervous system (Mauch et al., 2001). Cells internalize these particles and unesterified cholesterol and other lipids are trafficked from the endosomal-lysosomal system to organelles which they are destined, such as Golgi complex and endoplasmic reticulum (ER). In NPC cells, lipoprotein cholesterol particles are internalized without disruption, but stay entrapped in endosomal-lysosomal system, creating an insufficient efflux of these particles (Figure I.1). It leads to an accumulation of unesterified cholesterol, sphingolipids and complex gangliosides in cytoplasmic vesicles and a simultaneous scarcity of these lipids in organelles where they are required (Pacheco & Lieberman, 2008).

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Figure I.1. Lipid trafficking defects in NPC disease. Under normal conditions, cholesterol particles enter the

cell and are trafficked from endosomal-lysosomal system to the endoplasmic reticulum, Golgi complex and other intracellular organelles. In cells lacking NPC1 or NPC2, lipid trafficking is inhibited, leading to their accumulation in endosomes and lysosomes and no efflux to other intracellular compartments. Adapted from Pacheco & Lieberman, 2008.

The transport of sphingolipids from endosomes to Golgi complex can also be blocked by high levels of cholesterol (Vanier & Millat, 2003). Lower levels of cholesterol in the Golgi complex and ER result in deleterious effects in processes dependent on proper membrane composition and also in a scarcity of substrate for further synthetic reactions (Wojtanik & Liscum, 2003). Due to cholesterol sequestration, the subsequent induction of all low-density lipoprotein cholesterol-mediated homeostatic responses is retarded in NPC cells. Studies in patients cells demonstrated that lysosomal storage of unesterified cholesterol may show a changeable intensity; however, fibroblasts from a large amount of heterozygotes display mild but definitive changes (Argoff et al., 1991; Vanier et al., 1991). This impairment in the process of endocytosed cholesterol is essential for the pathogenesis of NPC disease and can clarify a more general dysfunction of intracellular lipid metabolism (Walkley & Vanier, 2009).

I-1.4. NPC genes and proteins

NPC disease is genetically heterogeneous, and it is possible to distinguish two complementation groups. Genes responsible for the disease have already been described. The NPC1 gene was identified in 1997, by positional cloning, as the gene mutated in the major complementation group (Carstea et al., 1997). This gene encodes a large membrane glycoprotein that is mainly localized in late endosomes-lysosomes

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19 (Higgins et al., 1999). In 2000, it was shown that the gene defective in the minor group was HE1/NPC2 (Naureckiene et al., 2000), which encodes a small soluble lysosomal protein with high affinity to cholesterol (Storch & Xu, 2009). Both genes are conserved during evolution, even in organisms in which cholesterol is not one of the most important components of membranes. The two genetic groups are biochemically and clinically impossible to differentiate and due to the resemblance in the disease phenotypes, it is believed that NPC1 and NPC2 may share the same metabolic pathway (Naureckiene et al., 2000; Sleat et al., 2004). Nonetheless, it was never verified any direct interaction between these two proteins (Ikonen & Holtta-Vuori, 2004).

The NPC1 gene, localized in chromosome 18q11-q12, encodes a 1278 amino acid integral membrane protein with 13 transmembrane domains. The NPC1 domain is a highly conserved region (amino acids 55-165) with a leucine zipper motif. The large cysteine-rich luminal loop (amino acids 855-1098) includes a ring-finger motif and is a likely site for protein-protein interaction. A sterol sensing domain (SSD) (amino-acids 615-797) displays high homology to SSD of other integral membrane proteins that act in response to ER cholesterol (Vanier & Millat, 2003) (Figure I.2).

Figure I.2. Topology of NPC1. LE/Lys – Late endosomes/Lysosomes. TM = Transmembrane region. Adapted from Lloyd-Evans & Platt, 2010.

The current number for identified NPC1 disease-causing mutations is close to 300, with a large majority of missense mutations, and more than 60 polymorphisms of the gene have also been described (Vanier, 2010). These missense mutations are scattered through the NPC1 gene and influence all functional domains, except the leucine zipper motif. While more than one-third of the mutations are located in the cysteine-rich luminal loop, there is a hot spot between amino acids 927 and 958, which harbors the three most frequent mutations (Vanier & Millat, 2003). The most common, in allele p.I1061T, is particularly frequent and is related with prominent cellular cholesterol trafficking disturbances in fibroblasts of patients and it is correlated with a juvenile neurological onset of NPC (Millat et al., 1999). The I1061T mutant was shown to be a functional protein targeted for endoplasmic reticulum-associated degradation (ERAD), due to protein

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misfolding (Gelsthorpe et al., 2008). Curiously, mutations corresponding to a less brutal impairment of cellular trafficking are located in this loop (Millat et al., 1999; Ribeiro et al., 2001; Sun et al., 2001).

Along with cysteine-rich luminal loop, SSD also reveals mutations that emphasized the functional significance of both domains. Homozygous mutations in SSD seem to be very deleterious, corresponding to a lack of mature NPC1 protein and to a very severe phenotype at both clinical and biochemical levels (Millat et al., 2001b).

The NPC2 gene, located in chromosome 14q24.3, is connected with very severe clinical phenotypes. Missense mutations in NPC2 have been associated to more diverse phenotypes, including juvenile and adult onset patients (Millat et al., 2001a; Verot et al., 2007). The mature NPC2 is a glycoprotein with a ubiquitous expression in several tissues (Naureckiene et al., 2000). Studies demonstrated a higher affinity binding and identified a hydrophobic cholesterol-binding pocket around amino acid K97 (Ko et al., 2003). There are few cases of NPC2 disease, but all present striking abnormalities of cellular cholesterol processing. It was suggested that NPC1 could be a regulator of NPC2 transport, but it was not confirmed. With the increase of NPC2 cases, it is clear that it has high heterogeneity as NPC1 (Vanier & Millat, 2003).

The exact functions of NPC1 and NPC2 have not yet been described (Vanier & Millat, 2003). The loss of function of both genes results in a versatile cellular pathology and, contrary to several other lipidoses that result from defects in enzyme activity, NPC seems to represent a primary transport defect. The failure of cholesterol homeostasis in NPC cells is known but whether the cholesterol transport defect is the main problem or potentially a consequence of some other malfunction remains unclear (Ikonen & Holtta-Vuori, 2004).

The majority of cell biological studies about NPC pathology are referent to cells defective in NPC1. NPC2 patients are rare and there are less models of study – a knock-out mouse model has only become available recently, contrary to NPC1 models, such as fibroblasts from affected patients, cells from the natural NPC1 -/- mouse (Loftus et al., 1997) and several cell lines in which NPC1 has been mutated (Millard et al., 2000). In the first studies, only biochemical assays were made concerning cholesterol trafficking, but actually there are already tools for morphological analysis of defective sterols, such as filipin staining (Coxey et al., 1993).

Some proteins related with cholesterol trafficking can also be involved in the generation of impairments in NPC cholesterol trafficking. ABCA1, a protein involved in the removal of cholesterol to apolipoprotein A-I, is suggested to resort to the endosomal-lysosomal pool to get cholesterol for its efflux (Chen et al., 2001). In fact, ABCA1-mediated cholesterol efflux is decreased in NPC1-deficient cells and the level of ABCA1

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21 protein is also lower in these cells. In contrast, sequestration of NPC1 in the cholesterol-laden late endocytic organelles in ABCA1-deficient (Tangier disease) fibroblasts was recently observed, suggesting that the functions of both proteins are associated (Neufeld et al., 2004).

The endosomal-lysosomal cholesterol sequestration in cells lacking NPC1 is paralleled by the failure of distribution of cholesterol to several intracellular compartments, such as the Golgi complex (Coxey et al., 1993), mitochondria (Frolov et al., 2003) and the ER (Neufeld et al., 1996). A defective cholesterol esterification and an impaired downregulation of cholesterol synthesis under cholesterol loading conditions indicate that probably there is a cholesterol deprivation in the ER of NPC cells (Brown & Goldstein, 1999; Liscum & Faust, 1987; Neufeld et al., 1996). Nevertheless, the analysis of cholesterol esterification in vitro using cell homogenates indicates that the ER cholesterol level in NPC cells is more or less normal, and the only impairment observed was the response to LDL-loading (Frolov et al., 2003; Lange et al., 2000).

The function of vesicular transport in endocytic cholesterol trafficking has been deeply studied and it is well established the role of endocytic pathway in the transport of LDL particles to organelles. The exit of cholesterol from late endosomes and lysosomes needs functional vesicular machinery, and vesicular trafficking defects are involved in NPC. Studies with green fluorescent protein (GFP)-fusion NPC1 protein in living fibroblast cultures have shown that this compartment undergoes rapid movements that are strikingly impaired in NPC1-mutant cells. These observations suggest that NPC1 is required for the production of tubulovesicular structures that show loss of flexibility and slower rate of movement in cells lacking NPC1. The NPC1-containing vesicles carry cholesterol from the perinuclear regions to the cell periphery. These structures were also observed to interact with the ER NPC1 (Ko et al., 2001; Zhang et al., 2001a).

It is possible to put forward two different scenarios concerning to vesicular transport and cholesterol accumulation: loss of NPC1 activity leads to an impaired motility and subsequently accumulation of cholesterol; instead, the accumulation of cholesterol accounts for the lack of motility – and it is a secondary defect. Since the available knowledge is limitative for conclusions, it seems plausible that both scenarios contribute to the phenotype (Ikonen & Holtta-Vuori, 2004).

Despite some ambiguous results, some experimental data suggest that NPC1 plays a role in regulation or mediation of retrograde transport of lysosomal cargo in the late endosomal-lysosomal pathway. NPC1 seems to be also an intervenient in transport or internalization of some compounds (Vanier & Millat, 2003).

It is believed that both NPC1 and NPC2 proteins function in a closely related fashion, since it was not found any qualitative difference in their ability to respond to exogenous

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22

LDL cholesterol loading and in their tissue lipid storage (Vanier et al., 1996). Ioannou (2001) suggested that NPC1 activity may be dependent on prior action of NPC2 to insert sterol into the endosomal-lysosomal membrane. However, more information about both proteins, such as structure and specific localization, may be useful to further understand these processes.

I-1.5. Conservation during evolution – NCR1, the yeast orthologue of hNPC1

NPC proteins are ubiquitously expressed and present homology with proteins in several organisms, indicating that NPC can play an important role in basic cellular processes. The yeast Saccharomyces cerevisiae has a single copy of NCR1, a NPC1 orthologue. Ncr1p contains multiple transmembrane domains, such as NPC1 domain, a conserved SSD domain, whose mutations highlight the importance of this domain for proper Ncr1p function (Malathi et al., 2004), and a cysteine-rich domain (Berger et al., 2005). Moreover, NPC1 proteins have an extremely high functional conservation among species. Indeed, Ncr1p is able to suppress cholesterol and ganglioside accumulation when expressed in NPC1-deficient Chinese hamster ovary cells (Malathi et al., 2004).

Many of the NPC1 patient mutations are in amino acids that are conserved in yeast proteins. Indeed, Ncr1p presents a rate of identity and similarity of 34% and 57%, respectively, when compared with human NPC1 (Carstea et al., 1997; Zhang et al., 2004). Furthermore, of 105 identified miscoding patient mutations, 66% of the affected amino acids are conserved in yeast and of these, 50% are identical between Ncr1p and human NPC1 (Berger et al., 2005).

The first phenotype related with NCR1 deletion in yeast was the resistance to the ether lipid drug, edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocoline). This resistance seems to be due to the inability to move the compound out of the vacuole, which probably provide protection to the cells from the inherent toxicity of edelfosine and allow cell growth in its presence. Despite all these features, previous work established that NCR1 is not essential for cell viability (Berger et al., 2005).

Other experiments were made to unravel the function of Ncr1p and subsequent relationship with NPC disease. Malathi and co-workers studied the effect of mutations in the SSD of Ncr1p and showed that Ncr1p plays an elemental role in subcellular sphingolipid distribution, by recycling sphingolipids, and that defects in this process result in sterols accumulation (Malathi et al., 2004). Despite these data, new studies are required to understand the role of Ncr1p at cellular levels.

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I-2. Endoplasmic reticulum stress

The endoplasmic reticulum (ER) is a specialized organelle with an important role in the biology of the cell (Guerin et al., 2008). This is one of the largest intracellular organelles, represented by a continuous membranous network that extends throughout the cytoplasm and is contiguous with the nucleus (Kaufman, 1999). In eukaryotic cells, the ER is the first compartment in the secretory pathway and the site of synthesis, folding and delivery of secreted, membrane-bound and organelle-targeted proteins that are correctly assembled to their proper targets within the secretory pathway and the extracellular space. Only proteins with a correct folding are exported to the Golgi complex, whereas incompletely folded proteins are retained in the ER to complete the folding procedure or marked for degradation, in a process called quality-control. The ER is the major site for synthesis of sterols and lipids and even a major part of the cell wall of lower eukaryotes is synthesized in the ER (Cid et al., 1995).

The ER provides the conditions required for protein folding, such as ATP, Ca2+ and an oxidizing environment that allows disulfide-bond formation and protein folding (Guerin et al., 2008; Shen et al., 2004). An appropriate ER function is essential for some cell physiological aspects, such as vesicle and lipid trafficking and protein targeting and secretion (Guerin et al., 2008). Environmental perturbations in these parameters, such as disruption of Ca2+ homeostasis, inhibition of protein glycosylation or disulfide bond formation, hypoxia and virus or bacteria infection, compromise the normal functioning of ER, leading to an accumulation and aggregation of unfolded proteins in the ER lumen that induces ER stress (Banhegyi et al., 2007; Shen et al., 2004). When the capability to process the protein folding is compromised, the ER activates a signal transduction pathway, known as unfolded protein response (UPR), in order to decrease the accumulation of these proteins in the ER (Kaneko & Nomura, 2003).

The UPR is the biochemical basis for several ER storage diseases, such as Huntington’s, Parkinson’s and Alzheimer’s disease, in which unfolded or misfolded proteins form aggregates (Rutishauser & Spiess, 2002; Vembar & Brodsky, 2008). This pathway was first described in the budding yeast S. cerevisiae (Back et al., 2005; Shen et al., 2004), after the identification of an unfolded protein response element (UPRE) in the yeast KAR2 promoter, by Sambrook and co-workers (Mori et al., 1993) and Walter and co-workers (Cox et al., 1993). The UPRE is essential and sufficient to confer ER stress inducibility on a heterologous reporter gene (Mori et al., 1992).

The ER has evolved mechanisms to sense the stress in the lumen and to induce an adaptive response that aims to reestablish its normal physiological state, either by up-regulating its folding capacity (by the induction of ER-resident molecular chaperones and

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foldases) and increasing its size, or down-regulating the biosynthetic load of the ER through shut-off of protein synthesis, at the transcriptional (Martinez & Chrispeels, 2003; Pakula et al., 2003) and translational level (Harding et al., 1999). In addition, the induction of ER associated degradation (ERAD) increases the degradation of unfolded proteins (Friedlander et al., 2000; Travers et al., 2000). When these mechanisms fail to restore normal ER homeostasis, apoptosis is activated to eliminate damaged cells (Shen et al., 2004).

I-2.1. Mechanism of protein folding

The ER holds specific characteristics, from its chemical composition to its machinery, that are different from those of other organelles and significantly influence protein folding processes. The ratio of glutathione forms (the major redox buffer in the cell) in the ER is also different from that in the cytosol. Levels of reduced (GSH) to oxidized glutathione (GSSG) in the ER is 1:1 to 3:1, against 30:1 to 100:1 in the cytosol (Hwang et al., 1992). The ER presents a neutral pH and high concentration of Ca2+ that can reach 5 mM, against 0.1 µM in the cytosol (Orrenius et al., 2003). Since the majority of ER-resident molecular chaperones and foldases have high affinity to Ca2+, perturbations of the ER Ca2+ pool can severely affect their folding and interactions with other chaperones (Corbett et al., 1999; Lloyd-Evans & Platt, 2010).

There are numerous post-translational alterations in the ER, such as disulfide bond formation and N-linked glycosylation. Glycosylation plays a key role in protein folding. This process starts at the ER, during protein synthesis in ribosomes and it is suggest that there is a higher thermodynamic stability of glycoproteins in the glycosylated form (Shental-Bechor & Levy, 2008). Disulfide bond formation is one of the most relevant parameters required for maturation of proteins in the ER. It is catalyzed by protein disulfide isomerases (PDI), which in turn is reoxidised by the FAD-dependent oxidase Ero1p. Ero1p is extremely important in yeast under anaerobic conditions and an uncoupling of Ero1p from its electron acceptor during ER stress, may lead to the formation of reactive oxygen species (ROS) (Tu & Weissman, 2002).

As a way to monitor if ER is assembling its products correctly, secreted proteins are targeted to ER quality control. Primary mediators of ER quality-control are molecular chaperones, which besides the sampling of correctly assembled proteins, help polypeptides to fold and evaluate the conformation of their substrates (Vembar & Brodsky, 2008). One of the ER quality-control machinery is the calnexin/calreticulin cycle, that analyzes protein conformations and defines if a molecule is exported to the Golgi complex or if is targeted for ERAD (Ellgaard et al., 1999).

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25 The protein folding machinery of the ER comprises three distinct groups of proteins: foldases, molecular chaperones and the lectins calnexin, calreticulin and EDEM (ER degradation-enhancing α-mannosidase-like protein). Foldases are enzymes whose role is to catalyze stages in protein folding. Chaperones increase the efficiency of protein folding by recognizing and stabilizing the partially folded intermediates during the folding process. They can be classified into several groups according to their cytosolic counterparts. One of these molecular chaperones is BiP, which takes part in the translocation of nascent polypeptide chains into the ER (Gupta & Tuteja, 2011). In fact, the interaction between unfolded proteins and ER-resident molecular chaperones represents a second quality-control checkpoint of the ER machinery (Ellgaard & Helenius, 2003).

I-2.2. Recognition of unfolded proteins

Biochemically, unfolded proteins present a conformation that interacts with molecular chaperones. However, different chaperones recognize and make possible the folding of different proteins. BiP also plays a role in recognition of unfolded proteins. This chaperone has high affinity to protein substrates. When unfolded proteins bind to BiP and become locked in their conformation, the ATPase activity of the chaperone is induced. BiP exists in equilibrium between monomeric and oligomeric forms. Only the monomeric form of BiP can bind unfolded proteins, and this association increases the monomeric, unmodified BiP pool (Freiden et al., 1992). Hence, it was suggested that BiP is recruited to the monomeric pool from a modified oligomeric BiP storage pool, by interaction with unfolded proteins (Gething, 1999). Furthermore, the UPR may respond to changes in the protein folding demand reflected by the available pool of free BiP (Shen et al., 2004). These mechanisms seem to be the first events in signal transduction, in response to the accumulation of unfolded proteins in the ER.

I-2.3. Transduction of the unfolded protein signal across the ER membrane

In higher eukaryotes there are three transmembrane proteins that transduce the unfolded protein signal across the ER membrane. Two of them belong to the ER luminal domains of the type I, IRE1 (inositol-requiring protein 1) and PERK (protein kinase RNA-like ER kinase), and the third is the type II transmembrane protein activating transcription factor 6 (ATF6) (Pineau & Ferreira, 2010; Shen et al., 2002). ATF6 holds two independent ER stress regulated Golgi localization sequences (GLS). Nonetheless, only PERK and IRE1 display a degree of conservation throughout all eukaryotes, and no ATF6 orthologue has been discovered in yeast until now (Liu et al., 2000; Torres-Quiroz et al., 2010).

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Experiments in yeast showed that the ER luminal domains of PERK and IRE1 are similar and their functions are evolutionarily conserved (Liu et al., 2000).

In an active state, there is a relationship between the luminal domains, PERK and IRE1, and BiP (Bertolotti et al., 2000). In fact, BiP can be considered as a master negative regulator of the UPR, because in unstressed cells, BiP binds to the luminal domains, keeping them inactive. However, when ER stress occurs and unfolded proteins accumulate in the lumen, BiP disassociates from these ER stress sensors to take part in protein folding attempt. Subsequently, oligomerization of luminal domains is initiated, as well as activation of these proximal signal transducers (Bertolotti et al., 2000; Shen et al., 2004).

The calnexin/calreticulin cycle and recognition of unfolded proteins by BiP play an important role in the regulation of activity of the proximal stress transducer ATF6 (Hong et al., 2004). Nevertheless, the conserved N-linked glycosylation site in yeast Ire1p is not essential for its function (Liu et al., 2000), which suggests that differential regulation of these three sections of UPR (IRE1, PERK and ATF6) exists to improve UPR signaling to specific folding requirements in the ER (Yoshida et al., 2003).

I-2.4. Activation of protective responses by the UPR – IRE1

Yeast and plants lack ATF6 and PERK. In yeast, the UPR is rather a simple linear pathway, with transcriptional regulation exclusively mediated by the IRE1 pathway, through the induction of chaperones and ERAD (Shen et al., 2004). This pathway is characterized by exclusive features in stress signal transduction and is observed in all eukaryotes. The IRE1 gene was identified in a forward genetic screen for mutations related with the activation of an UPRE::LACZ reporter by ER stress (Cox et al., 1993; Mori et al., 1993). IRE1 encodes a type I transmembrane ER resident protein, with an N-terminal luminal domain that senses the ER stress and a C-N-terminal cytoplasmic domain required for KAR2 (the yeast orthologue of BiP) expression (Shen et al., 2004).

The substrate for the Ire1p endoribonuclease is the mRNA for the bZIP transcription factor Hac1p. HAC1 contains a large intron of 252 bp, located in the 3’-end of the mRNA. The presence of unfolded proteins in the ER lumen, induced by agents such as tunicamycin, a natural inhibitor of N-linked glycosylation, or dithiothreitol (DTT), which impairs disulfide bond formation, leads to the dimerization and trans-autophosphorylation of Ire1p (Back et al., 2005; Fei et al., 2009; Shen et al., 2004). This activates its RNase activity and induces the cleavage of both 5’- and 3’-exon-intron junctions in HAC1 mRNA, leading to the formation of 5’-OH and 3’-cyclic PO4 ends (exons), that are joined by tRNA

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27 was first described in yeast as an immediate downstream substrate for the RNase activity of Ire1p, and showed ability to bind with KAR2 UPRE (Mori et al., 1996). Notwithstanding, there are no readily recognizable UPREs in the promoters of the genes associated with ERAD (Back et al., 2005).

The mechanism of HAC1 mRNA splicing is similar to pre-tRNA splicing, but differs in the localization, since HAC1 mRNA splicing is likely to be cytoplasmic. Despite the resemblance of both splicing mechanisms, it is unknown how the ligase differentiates exons and introns. In vitro assays showed that HAC1 exons remain associated after the cleavage induced by Ire1p (Abelson et al., 1998; Gonzalez et al., 1999). The translation of unspliced mRNA is suppressed by the base pairing between the 5’-UTR (untranslated region) of unspliced HAC1 mRNA and the intron (Ruegsegger et al., 2001). However, the increased transcriptional activation potential that is observed in the spliced forms opposed to the unspliced ones is not yet fully explained.

The HAC1 mRNA splicing leads to the expression of an alternative C-terminus with high transcriptional activation potential and to the removal of a translational attenuator from HAC1 mRNA (Mori et al., 2000; Ruegsegger et al., 2001). Then, Hac1p binds to the UPRE (CAGCGTG) (Mori et al., 1998). After suppression of protein synthesis in ribosomes, ER chaperones are induced to correct protein conformation, by refolding unfolded proteins. The remaining unfolded proteins are then eliminated from the ER to the cytosol through retrograde transport, and degraded by the proteasome (ERAD) (Kaneko & Nomura, 2003).

Figure I.3. The Ire1p cascade of the UPR pathway. Ire1p detects high levels of misfolded proteins in the ER

lumen and promotes HAC1 mRNA splicing reaction, removing the intron from the precursor mRNA which encodes Hac1p. Hac1p binds to UPR related genes and upregulate the expression of chaperones and ERAD proteins in the nucleus.

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I-2.5. Role of IRE1 and HAC1 in membrane proliferation control

The UPR may have a role in the regulation of membrane proliferation. In cells lacking Ire1p and Hac1p (that are inositol auxotrophs), ER stress triggered by tunicamycin (TUN) upregulate INO1 gene, which encodes for inositol-1-phosphate synthase, a key enzyme in phospholipid biosynthesis (Cox et al., 1997; Travers et al., 2000). In addition, the induction of membrane proliferation is in some cases dependent on a functional UPR pathway (Cox et al., 1997; Takewaka et al., 1999). Therefore, UPR seems to hold a specialized function in the increment of phospholipid biosynthesis and ER proliferation when it comes to an acute and/or severe ER stress. On the other hand, studies with ire1Δ and hac1Δ mutants revealed that the activation of INO1 by inositol starvation was only moderately defective in these strains (Chang et al., 2002; Cox et al., 1997). These mutant strains presented, after 4h inositol starvation, increased values of CDP-diacylglycerol, compared to wild-type, and decreased levels of phosphatidic acid and phosphatidylinositol. These alterations were reversed and INO1 induction was not compromised by HAC1 deletion in a strain with an overexpression of inositol phenotype (Opi-). The changes in phospholipid levels in ire1Δ and hac1Δ strains indicate that UPR plays a role in the regulation of metabolic reactions in phospholipid metabolism at the ER membrane (Chang et al., 2002).

The bZIP transcription factor downstream of IRE1 presents a high degree of divergence, even in organisms evolutionarily close, such as yeasts and filamentous fungi (Saloheimo et al., 2003). In metazoans, XBP1 (X-box binding protein-1) is a bZIP transcription factor of the ATF/CREB family and is the functional homologue for Hac1p. It plays a key role in the regulation of a subset of ER-resident molecular chaperones. XBP1 splicing also introduces a frame-shift and an alternative C-terminus with increased transcriptional activation potential. The mechanism of XBP1 splicing is still unclear. Nevertheless, despite the divergence, the splice junctions in both XBP1 and HAC1 mRNA are conserved (Lee et al., 2002).

Being the only major pathway in yeast, the IRE1 pathway coordinates several features of the secretory pathway, such as membrane biogenesis, chaperone induction, upregulation of ERAD genes and ER quality-control (Friedlander et al., 2000; Travers et al., 2000; Yoshida et al., 2003). Previous experiments have shown that moderate IRE1- and HAC1-independent transcriptional induction from a core promoter occurs in response to ER stress in yeast. Thus, a second signal transduction pathway that modulates and stimulates activation of ER chaperone genes by a Ire1p-hac1p independent pathway, in response to stress, may exist in this organism (Schroder et al., 2003).

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I-2.6. Endoplasmic reticulum-associated degradation (ERAD)

ER stress conditions can also induce the proteasome-dependent ERAD system to counteract the high levels of unfolded or misfolded proteins present in the lumen of the ER and restore ER homeostasis (Guerin et al., 2008).

ERAD systems are ubiquitous among eukaryotes and have been well studied in S. cerevisiae (Xie & Ng, 2010). These systems aim to remove unfolded proteins by retrograde transport from the ER to the cytosol via the translocon and, consequently, sort them for degradation by the ubiquitin-proteasome system (UPS) (Kaneko & Nomura, 2003). Unfolded proteins in the lumen of the ER are recognized through the detection of specific domains, such as unpaired cysteines or exposed hydrophobic regions. Retrotranslocation of these proteins from the ER to the cytoplasm may use the same core protein complex Sec61p that provides the conducting channel in the translocon through which proteins are imported into the ER lumen. Then, a cascade of enzymatic reactions leads to a formation of a polyubiquitinated protein that will be recognized by the proteasome subunits and subsequently degraded. Initially, the UPS was connected with an ER quality control mechanism. Several studies indicated later that the UPS is able to degrade proteins with anomalous conformations, leading to discover of some proteins that are targeted to degradation by UPS, such as the yeast vacuolar protease carboxypeptidase Y (CPY*), which is unfolded, retained and later degraded by ERAD (Vembar & Brodsky, 2008; Xie & Ng, 2010).

In yeast, many components of ERAD pathway are induced by the UPR, such as DER1, HRD3, HRD1/DER3 and UBC7 (Travers et al., 2000). Hrd1p is an ER type I transmembrane protein that has E2 ubiquitin ligase activity (Bays et al., 2001); nevertheless, it prefers an unfolded protein as an ubiquitination substrate and uses only E2 ubiquitin-conjugating enzymes to mediate ubiquitination of ERAD substrates. Although UBC1 mRNA levels are not affected by DTT treatment, UBC7 and HRD1 genes are induced upon ER stress by a Hac1p- and Ire1p-dependent mechanism, implying that the UPR may regulate some parts of ERAD system in yeast. The UPR is not essential for basal expression of these proteins, suggesting that there is a basal level of ERAD, sufficient for elimination of unfolded proteins under normal physiological conditions. However, upon ER stress, the UPR is induced to increase ERAD activity to face the new conditions (Friedlander et al., 2000).

The association between UPR and ERAD is not totally understood. It is known that cells unable to perform ERAD are more sensitive to stress, as observed by a constitutive activation of the UPR and a requirement for the UPR for normal growth and survival under

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mild stress conditions (Friedlander et al., 2000). Some unfolded protein substrates of the ERAD pathway were characterized in cells lacking IRE1 (Casagrande et al., 2000). The identification in mammalian cells of EDEM also supports the idea of an interaction between the UPR and ERAD. The induction of EDEM on ER stress conditions is mediated by IRE1/XBP1, but not by ATF6, suggesting that one of the functions of IRE1/XBP1 is to upregulate ERAD (Yoshida et al., 2003).

I-2.7. Cell signaling pathways related with ER stress conditions – the High Osmolarity Glycerol (HOG) pathway

When environmental conditions change, organisms evolve responses in order to survive that include the induction of cell signaling pathways. These comprise Mitogen-Activated Protein Kinase (MAPK) cascades that consist of a three component signaling system, namely a MAPK Kinase Kinase, a MAPK Kinase and a MAPK that are sequentially activated by phosphorylation (Robinson & Cobb, 1997).

Many of these MAPK cascades are evolutionarily conserved in eukaryotes (Chen & Thorner, 2007). In S. cerevisiae, there are five MAPK pathways: pheromone response pathway mediates cellular responses to pheromones; filamentous growth pathway leads to a regulation to nutrient limiting conditions; spore wall assembly pathway acts during meiosis and sporulation; cell wall integrity pathway is involved in conditions of cell wall stress, such as hypo-osmotic shock; and the High Osmolarity Glycerol (HOG) pathway plays a key role in survival under hyperosmotic conditions.

The HOG pathway is activated in response to an increase of osmolarity in extracellular medium, leading to higher glycerol production. In order to maintain osmotic balance, cells also increase glycerol uptake and, therefore, intracellular osmolyte concentration. Several studies indicate the HOG pathway as an essential cascade for regulating adaptation to severe conditions, such as heat stress and citric acid. This pathway consists of two branches that encompass putative osmosensors coupled to a MAPK cascade that, by phosphorylation, may lead to the activation of the Hog1p MAPK, the orthologue to mammalian p38 stress-activated protein kinase (SAPK) (Schroeter et al., 2002; Torres-Quiroz et al., 2010) (Figure I.4).

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Figure I.4. The High Osmolarity Glycerol (HOG) pathway. During osmotic stress, the HOG pathway is

induced and the Hog1p MAPK phosphorylates and activates transcription factors that mediate stress response.

It was recently shown that the HOG pathway also has a major role in ER stress resistance. The mechanism of Hog1p phosphorylation during ER stress is divergent from that associated with cellular response to other stress conditions and only Sln1p branch of the HOG pathway is required, along with both Ire1p and Hac1p (Bicknell et al., 2010; Torres-Quiroz et al., 2010). Strains lacking Hog1p present sensitivity to tunicamycin or β-mercaptoethanol (Torres-Quiroz et al., 2010), a reducing agent that also induces ER stress by preventing disulfide bond formation. These results indicate that Hog1p is vital to deal with chemical agents that form unfolded protein aggregates in the ER. In contrast, when a hyperactivation of this pathway occurs, cells reveal resistance to tunicamycin, indicating that kinase activity of Hog1p is necessary to deal with N-glycosylation defects promoted by tunicamycin exposure (Torres-Quiroz et al., 2010).

The HOG pathway is also involved in late phases of ER stress (Bicknell et al., 2010). Hog1p translocates into the nucleus and controls the expression of genes that are exclusively activated in late points of ER stress. Hog1p also induces autophagy components (Prick et al., 2006), indicating that the HOG pathway takes part in several aspects of cellular response to long term ER stress.

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I-3. Saccharomyces cerevisiae as biological model

The yeast Saccharomyces cerevisiae is a unicellular eukaryotic fungus encompassed in the Ascomycete family. It is frequently used as a model organism for the study of the eukaryotic cell and biological processes conserved during evolution (Jazwinski, 2005). It is established that S. cerevisiae is appropriate to the study of fundamental cellular mechanisms and correlations with those in higher eukaryotes, including humans. Due to the high degree of similarity among eukaryotes – from the organization and function of molecules, organelles, genes, to signaling pathways necessary for the regulation of cell growth (Botstein et al., 1997), stress responses (Gasch & Werner-Washburne, 2002) and intracellular transport (Kucharczyk & Rytka, 2001) – it is possible to study all of these mechanisms in such a simple organism as yeast.

S. cerevisiae was the first organism to have its genome fully sequenced and a major part is functionally characterized. Indeed, the development of genomic and proteomic tools, combined with the several online databases that contain information about yeast genes and proteins (Pena-Castillo & Hughes, 2007), provides a wide range of knowledge about several aspects of the organism. It is also a microorganism easy to genetically manipulate and techniques for its manipulation and harvesting are strongly optimized (Amberg et al., 2005).

Since yeast presents orthologues of human genes, it has been used for the study of several diseases, including cancer (Hartwell, 2002) and neurological disorders like Huntington’s (Giorgini et al., 2005). S. cerevisiae has also been used as a model for the study of NPC disease, due to the existence of hNPC1 and hNPC2 orthologues, NCR1 and NPC2, respectively. In fact, the identification of a phenotype for the ncr1Δ mutants allows the use of conventional yeast genetics to define cell functions for NPC proteins (Berger et al., 2005). It is also possible to use yeast to study mechanisms related with ER stress response, due to the conservation during evolution of the IRE1 pathway, which controls UPR, and its downstream target, the Hac1p transcription factor (orthologue of human XBP1) (Shen et al., 2004). Therefore, we selected S. cerevisiae as our model organism to study the role of Ncr1p in ER stress response.

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Chapter II

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35 Previous studies propose neurodegenerative diseases as one of the main causes of accumulation of unfolded proteins and subsequent ER stress (Kaneko & Nomura, 2003). Thereby, we decided to unravel the role of Ncr1p in endoplasmic reticulum stress response in S. cerevisiae. This work aimed to:

i. characterize the sensitivity of ncr1Δ cells to stress conditions, by exposure to compounds that induce ER stress;

ii. establish the correlation between ER stress sensitivity and oxidative stress markers, by measuring ROS and glutathione levels;

iii. uncover putative alterations in cell signaling pathways of ncr1Δ cells, related to ER stress response

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Chapter III

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" Role Of In Endoplasmic Reticulum Stress Response In Sacccharoromyces cerevisiae ".

ROLE OF Ncr1p IN ENDOPLASMIC RETICULUM STRESS

RESPONSE IN Saccharomyces cerevisiae

JOANA FILIPA MADUREIRA GAIFEM

Dissertação de Mestrado em Contaminação e Toxicologia Ambientais

JOANA FILIPA MADUREIRA GAIFEM

ROLE OF Ncr1p IN ENDOPLASMIC RETICULUM STRESS

RESPONSE IN Saccharomyces cerevisiae

Dissertação de Candidatura ao grau de

Mestre em Contaminação e Toxicologia

Ambientais submetida ao Instituto de

Ciências Biomédicas de Abel Salazar da

Universidade do Porto.

Orientador – Doutor Vítor Costa

Categoria – Professor Associado

Afiliação

– Instituto de Biologia Molecular e

Celular; Instituto de Ciências Biomédicas de

Abel Salazar

Acknowledgements

Table of Contents

Figure Index

Table Index

Abbreviations

Abstract

Resumo

Chapter I

I-1. Niemann-Pick type C disease

I-2. Endoplasmic reticulum stress

I-3. Saccharomyces cerevisiae as biological model

Chapter II

Chapter III