Tesis sobre el tema "Endoplasmic Reticulum"

Secretory proteins that are unable to assemble into native proteins in the endoplasmic reticulum (ER) are transported back into the cytosol for degradation. Many cytosolic and ER resident proteins have been identified so far as being involved in this retrotranslocation process, but it is difficult to determine whether these proteins have a direct or indirect effect. Interpretations are further complicated if the loss of a specific protein is obscured by the presence of another protein that is partially or wholly redundant. To overcome these limitations, a mammalian in vitro system was developed that allowed to monitor retrotranslocation synchronously and in real time in the absence of concurrent translocation. To examine the roles of different components in ER-associated degradation (ERAD), well-defined and homogeneous mammalian ER microsomes were prepared biochemically by encapsulating a fluorescent-labeled ERAD substrate with specific lumenal components. After mixing ATP, specific cytosolic proteins, and specific fluorescence quenching agents with microsomes, substrate retrotranslocation was initiated. The rate of substrate efflux from microsomes was monitored spectroscopically and continuously in real time by the reduction in fluorescence intensity as the fluorescent substrates passed through the ER membrane and were exposed to the quenching agents. Retrotranslocation kinetics were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase, or all cytosolic proteins with only the 19S proteasome cap. Retrotranslocation was blocked by affinity-purified antibodies against Derlin1, but not by affinity-purified antibodies against Sec61α or by membrane-bound ribosomes. Since the substrate also photocrosslinked Derlin1, but not Sec61α or TRAM, retrotranslocation of this ERAD substrate apparently involves Derlin1, but not the translocon. By labeling either the C- or N-terminus, it was revealed that the N-terminus of one ERAD substrate leaves the ER lumen first. This finding suggests that the protein is retrotranslocated as a linear polymer in a preferred direction. When RRMs were reconstituted with a fluorescent-labeled ERAD substrate and various ions. Ca2+ ions in the ER lumen increased the rate and extent of retrotranslocation, while Ca2+ ions in the cytosol decreased retrotranslocation. This approach therefore provides the first direct evidence of the involvement and importance of specific ionic requirements for ERAD.

One of the challenges of quantitative approaches to biological sciences is the lack of understanding of the interplay between form and function. Each cell is full of complex-shaped objects, which moreover change their form over time. To address this issue, we exploit recent advances in confocal microscopy, by using data collected from a series of optical sections taken at short regular intervals along the optical axis to reconstruct the Endoplasmic Reticulum (ER) in 3D, obtain its skeleton, then associate to each of its edges key geometric and dynamic characteristics obtained from the original filled in ER specimen. These properties include the total length, surface area, and volume of the ER specimen, as well as the length surface area, and volume of each of its branches. In a view to benefit from the well established graph theory algorithms, we abstract the obtained skeleton by a mathematical entity that is a graph. We achieve this by replacing the inner points in each edge in the skeleton by the line segment connecting its end points. We then attach to this graph the ER geometric properties as weights, allowing therefore a more precise quantitative characterisation, by thinning the filled in ER to its essential features. The graph plays a major role in this study and is the final and most abstract quantification of the ER. One of its advantages is that it serves as a geometric invariant, both in static and dynamic samples. Moreover, graph theoretic features, such as the number of vertices and their degrees, and the number of edges and their lengths are robust against different kinds of small perturbations. We propose a methodology to associate parameters such as surface areas and volumes to its individual edges and monitor their variations with time. One of the main contributions of this thesis is the use of the skeleton of the ER to analyse the trajectories of moving junctions using confocal digital videos. We report that the ER could be modeled by a network of connected cylinders (0.87μm±0.36 in diameter) with a majority of 3-way junctions. The average length, surface area and volume of an ER branch are found to be 2.78±2.04μm, 7.53±5.59μm2 and 1.81±1.86μm3 respectively. Using the analysis of variance technique we found that there are no significant differences in four different locations across the cell at 0.05 significance level. The apparent movement of the junctions in the plant ER consists of different types, namely: (a) the extension and shrinkage of tubules, and (b) the closing and opening of loops. The average velocity of a junction is found to be 0.25μm/sec±0.23 and lies in the range 0 to 1.7μm/sec which matches the reported actin filament range.

The mammalian endoplasmic reticulum (ER) is the largest organelle in the cell, extending from the nuclear envelope throughout the cell periphery. The ER houses a wide variety of vital cell processes within a single membrane bound organelle. In order to accommodate these functions and respond to the demands of the cell, the ER is partitioned into dynamically regulated subdomains, each with its own distinct structure. Despite the likely importance of ER structure for its functions, few proteins have been identified as having a direct role in maintaining the structure of the ER and the consequences of alteration of normal ER structure are not well understood. Here we identify Yip1A, a conserved membrane protein that cycles between the ER and early Golgi, as a likely regulator of ER organization. Yip1A depletion led to restructuring of ER membranes into micrometer-sized, concentrically stacked whorls. These structures are reminiscent of the ER whorls found in certain specialized secretory cell types, where the regulation and functional consequence of ER whorl formation is not understood. We found that membrane stacking and whorl formation after Yip1A depletion coincided with a marked slowing of coat protein (COP) II-mediated protein export from the ER. Furthermore, whorl formation driven by exogenous expression of an ER protein with no role in COPII function also delayed cargo export. Thus, it appears that Yip1A is required to prevent ER whorl formation and that whorl formation can in turn delay protein export from the organelle. Whether this is the function of ER whorls in tissues remains to be seen, however these results make Yip1A a good candidate for playing a role in their regulation. To obtain insight into how Yip1A regulates ER whorl formation and to determine whether the mechanism might be shared with the yeast homologue Yip1p, we carried out a systematic mutational analysis of all residues in the protein. Two discrete sites (E95 and K146) were crucial for the control of ER whorl formation by Yip1A. Notably, the same residues were previously shown to be important for Yip1p-mediated viability in yeast, indicating a shared mechanism. On the other hand, a third site (E89) also essential for yeast viability was dispensable for Yip1A function in regulating whorl formation. Thus Yip1p/Yip1A may possess at least two distinct essential functions only one of which is required for regulation of ER structure. Of note, the sites required for control of ER whorl formation by Yip1A were dispensable for the binding of Yip1p to its established binding partners Yif1p and Ypt1/31p, whereas the site required for Yip1p to bind the same partners was dispensable for ER structuring by Yip1A. Based on these observations, we speculate that the function of Yip1A in regulating whorl formation is mediated by one or more distinct and yet-to-be identified binding partners. Collectively, these findings indicate that a dispersed ER network is important for proper COPII-mediated protein export and that Yip1A has a conserved function between yeast and humans in maintaining proper ER network dispersal through prevention of ER whorl formation. These studies set up an important framework for determining the molecular mechanism of Yip1A as an ER structuring protein

Of the membrane bound organelles in eukaryotic cells, the endoplasmic reticulum (ER) may be the most complex. It is the largest both in terms of surface area and volume. It includes several subdomains: the nuclear envelope (NE) as well as an extensive network of both highly inter-connected fenestrated tubular membranes and flat cisternal sheets (1, 2). While the structure and organization of the ER is thought to be important for the execution of a myriad of essential cellular functions including protein and lipid synthesis and export as well as calcium sequestration and drug metabolism (3), how this membrane system is generated and maintained despite continuous turnover is only just beginning to be unraveled (4). The microtubule cytoskeleton and molecular motors are clearly important for the extension of tubules from the existing network so that they may fuse with nearby ER tubules to generate new three-way junctions (5-8). However, an ER-like network can be generated in vitro in the absence of microtubules (9), which suggests the existence of additional mechanisms for the extension and scaffolding of the ER network. Once the tubular ER does extend out from the existing network the next critical step is fusion. Soluble NSF attachment-protein receptors (SNAREs) were an obvious candidate for this role (10), but ER homotypic fusion events have not yet been found to depend on SNAREs. Recently, a member of the dynamin super-family of large GTPases, atlastin, was implicated in ER homotypic fusion. An in vitro fusion assay (11) and knockdown experiments (12) in conjunction with the crystal structures of the soluble domains of atlastin (13, 14) have led to a possible mechanism of ER fusion, but this model remains to be tested. In my thesis I will describe two projects. One focuses on the role and mechanism of nucleoside diphosphate kinase B (NDKB) in ER network extension and stabilization. The other focuses on the role and mechanism of atlastin in fusing ER membranes. NDKB was initially implicated as a stimulator of ER export in permeabilized cells (15). Subsequent work suggested that its effect on ER export might be through an effect on ER network morphology. Through in vitro assays, we found that NDKB not only stabilizes the ER network but also actively promotes ER network extension. In order to perform this function we hypothesized that it might interact directly with ER membranes. Indeed, we found a pair of positively charged residues that mediated direct binding of NDKB to anionic phospholipids. When these residues ware mutated to negatively charged residues, NDKB no longer bound anionic phospholipids and failed to mediate ER extension in our semi-intact cell assay. In order to gain insight into the mechanism for how NDKB might be performing its ER network extension function we took another in vitro approach. Anionic synthetic liposomes were incubated with NDKB and we found that NDKB was able to arrange these liposomes into large arrays that resembled the ER network. Together these results implicate NDKB and anionic phospholipids in a role for ER network morphology, in particular as a means to stabilize and extend the ER network. We initially became interested in the atlastin GTPase as a result of an ER overexpression phenotype observed by the Blackstone lab indicating a potential role in ER morphogenesis (16). In strong support for a required role for atlastin in ER structuring, we found that siRNA depletion of atlastin from HeLa cells resulted in a reduction in network density which could be rescued by the addition of an siRNA immune atlastin transgene. This established a structure function assay we could use to dissect the functional domains of atlastin. Concurrent with our identification of key residues required for atlastin function, it was observed by another lab that atlastin could fuse synthetic liposomes (11), suggesting that the ER structuring role we had observed for atlastin might correspond to the membrane fusion step. Simultaneously, structure determinations for the soluble domain of atlastin were reported (13, 14). Together, the collective data suggested the following model for atlastin: GTP dependent dimerization of atlastin leads to tethering and subsequent GTP hydrolysis leads to a large conformational change that drives membrane fusion (13, 14). To test the model, we exploited our identification of a required salt bridge central to the large conformational change proposed to convert GTP-bound tethered atlastin dimers into a postfusion state. We established that although blocking the salt bridge had no effect on GTP binding and hydrolysis, it abolished stable atlastin dimer formation. Then, through a series of crosslinking assays probing the conformational state of the atlastin soluble domain, we showed that atlastin adopts the postfusion conformation in the GTP bound state, without the need for GTP hydrolysis. As a result of our studies, we have modified the current model for atlastin’s function. In our revised model, GTP binding is required for atlastin’s initial dimerization and begins a cascade of conformational changes that results in the large rearrangement thought to drive membrane fusion. We speculate based on our work that hydrolysis may be necessary to complete the fusion cycle and/or function to disassemble the postfusion complex for multiple rounds of fusion. In summary, these studies provide both an initial analysis of a protein with an ER network extension role and important insights into the mechanism of ER membrane fusion. It is hoped that this work will add to our understanding of the biogenesis and maintenance of the ER network.

The biosynthesis of integral membrane proteins (IMPs) is an essential cellular process. IMPs comprise roughly 20-30% of the protein coding genes of all organisms, nearly all of which are inserted and assembled at the endoplasmic reticulum (ER). The defining structural feature of IMPs is one or more transmembrane domains (TMDs). TMDs are typically stretches of predominately hydrophobic amino acids that span the lipid bilayer of biological membranes as an alpha helix. TMDs are remarkably diverse in terms of their topological and biophysical properties. In order to accommodate this diversity, the cell has evolved different sets of machinery that cater to particular subsets of proteins. Our knowledge of how the TMDs of IMPs are selectively recognized, chaperoned into the lipid bilayer, and assembled remains incomplete. This thesis is broadly interested in investigating how TMDs are correctly inserted and assembled at the ER. To address this the biosynthesis of multi-pass IMPs was first considered. Multi-pass IMPs contain two to more than twenty TMDs, with TMDs that vary dramatically in terms of their biophysical properties such as hydrophobicity, length, and helical propensity. The beta-1 adrenergic receptor (β1-AR), a member of the G-protein-coupled receptor (GPCR) family was established as a model substrate in an in vitro system where the insertion and folding of its TMDs could be interrogated. Assembly of β1-AR is not a straightforward process, and current models of insertion fail to explain how the known translocation machinery correctly identifies, inserts, and assembles β1-AR TMDs. An in vivo screen in mammalian cells was therefore conducted to identify additional factors which may be important for multi-pass IMP assembly. The ER membrane protein complex (EMC), a well conserved ER-resident complex of unknown biochemical function, was identified as a promising hit potentially involved in this assembly process. The complexity of working with multi-pass IMPs in an in vitro system prompted the investigation of a simpler class of proteins. Tail-anchored proteins (TA) are characterized by a single C-terminal hydrophobic domain that anchors them into membranes. Though structurally simpler compared to multi-pass IMPs, the TMDs of TA proteins are similarly diverse. We found that known TA insertion pathways fail to engage low-to-moderately hydrophobic TMDs. Instead, these are chaperoned in the cytosol by calmodulin (CaM). Transient release from CaM allows substrates to sample the ER, where resident machinery mediates the insertion reaction. The EMC was shown to be necessary for the insertion of these substrates both in vivo and in vitro. Purified EMC in synthetic liposomes catalysed insertion of its TA substrates in a fully reconstituted system to near-native levels. Therefore, the EMC was rigorously established as a TMD insertase. This key functional insight may explain its critical role in the assembly of multi- pass IMPs – which is now amenable to biochemical dissection.

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity-related inflammation and insulin resistance in adipose tissue. However, the mechanisms responsible for induction of ER stress are presently unclear. Proper ER redox state is crucial for oxidative protein folding and secretion and impaired protein folding in ER leads to induction of unfolded protein response and ER stress. However, while ER redox state is more oxidizing compared to the rest of the cell, its regulation is poorly understood. In order to determine the effects of ER redox state on development of ER stress and insulin resistance, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. In this study we attempted to develop and characterize a sensitive tool to study the ER redox state in adipocytes in real-time by targeting a new generation of redox-sensitive green fluorescent protein (roGFP) to ER. The roGFP1-iL sensor targeted to the ER is termed ‘eroGFP1-iL’ by convention. The ER-targeting eroGFP1-iL construct contains the signal peptide from adiponectin and the ER retention motif KDEL and has a midpoint reduction potential of -229 mV in vitro in oxidized and reduced lipoic acid. Despite having a midpoint reduction potential that is 50 mV higher than the previously determined midpoint reduction potential of the ER, eroGFP1-iL was found capable of detecting both oxidizing and reducing changes in the ER. In an attempt to determine the mechanisms by which roGFP1-iL detects oxidizing changes, we found that, first, glutathione mediated the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitated dimerization of roGFP1-iL, which in effect shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule’s reduction potential compared with a dithiol redox buffer like lipoic acid. From this study, we concluded that the glutathione redox couple in ER significantly raised the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in ER with high reliability in real-time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggest a range of ER redox potential consistent with those determined by earlier approaches that did not involve fluorescent sensors. Our primary aim in developing eroGFP1-iL as a redox-sensing tool was to be able to assess whether redox changes represent an early initiator of ER stress in obesity-induced reduction in high molecular weight (HMW) adiponectin in circulation. Hypoxia is a known mediator of redox changes. We found that oligomerization of HMW adiponectin was impaired in the hypoxic conditions observed in differentiated fat cells. The redox-active antioxidant ascorbate was found capable of reversing hypoxia-induced ER stress. Lastly, we demonstrated that changes in ER redox condition is associated with ER stress response and is implicated in the mechanism of action of the insulin-sensitizing agent troglitazone and desensitizing agent palmitate. Using the redox sensing property of eroGFP1-iL, palmitate was found to be an effective modulator of redox changes in the ER and troglitazone was found to cause oxidizing changes in the ER. The action of palmitate in causing aberrant ER redox conditions was associated with aberrant HMW adiponectin multimerization. Palmitate-induced ER stress was ameliorated by troglitazone. Taken together, the data suggest a potential role of ER redox changes in ER stress and impaired protein folding in adipocytes.

Protein-protein interactions in the endoplasmic reticulum (ER) are essential for concerted action of higher molecular complexes. However, the presence of transmembrane domains (TMDs) in many ER proteins and the environmental conditions of the ER prohibit protein-protein interactions from being identified in the ER. To detect interactions between membrane and luminal proteins of the ER, the ER Membrane Yeast Two-Hybrid (MYTH) system uses the ER type 1 membrane protein Ire1p as a reporter of protein interactions in the ER. A hyperactive Ire1p system was developed to determine the C-terminal topologies of ER and ER-related proteins to generate functional N-terminal fusions to Ire1p for the MYTH system. Among 256 proteins, which were hard to analyze on a high-throughput basis by other approaches, 454 interactions were identified. Results showed novel links between previously established biological processes, such as that between the SRP-dependent and independent pathways, and provided roles for ER and ER-related proteins.
Les interactions protéine-protéine dans le réticulum endoplasmique (RE) sont essentielles pour qu'une action concertée des complexes moléculaires de haut poids ait lieu. Cependant, la présence de domaines transmembranaires dans plusieurs protéines et les conditions environnementales du RE ne permettent pas l'identification des interactions protéine-protéine dans cette organite. Afin de détecter les interactions entre les protéines membranaires et luminales du RE, nous avons utilisé la technique du MYTHS (Membrane Yeast Two-Hybrid System) qui emploie la protéine membranaire de type 1, Ire1p, comme rapporteur des interactions des protéines dans le RE. Un système du Ire1p hyperactif a été développé afin de déterminer la topologie de l'extrémité C-terminale des protéines du RE ainsi que celles qui sont y reliées en les fusionnant à l'extrémité N-terminale du Ire1p du MYTHS. Parmi les 256 protéines qui étaient difficiles à analyser par d'autres méthodes à haut débit, 454 interactions sont identifiées. Les résultats démontrent des nouveaux liens entre les fonctions biologiques déjà établies comme celles entre les voies dépendantes et indépendantes de la particule de reconnaissance du signal (SRP) et postulent des rôles aux protéines du RE ainsi qu'à celles qui y sont reliées.

Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.
Cataloged from PDF version of thesis. "February, 2011."
Includes bibliographical references.
Quality control is an important part of protein biogenesis. Aberrant proteins must be destroyed before they aggregate and cause deleterious effects. Failure to do so can result in cell death or malfunction and, ultimately, disease. Quality control involves the recognition of misfolded proteins and their degradation. For secretory and membrane proteins, folding occurs in the endoplasmic reticulum (ER), but degradation is performed by the cytosolic proteasome. Thus, quality control in the secretory system includes the dislocation of misfolded proteins from the ER to the cytoplasm. ER proteins that fail to fold correctly are identified and directed to the membrane-associated ER quality control machinery. They are then moved across the ER membrane, tagged with ubiquitin, and extracted from the membrane and complex. Finally, the dislocation substrate is taken to the proteasome where it is degraded. Many proteins constituting the mammalian quality control machinery have been identified. Several different dislocation complexes work in parallel to clear misfolded proteins from the ER and they are distinguished by their E3 ubiquitin ligase. This thesis describes the identification and characterization of several previously unknown members of the HRD1-associated ER quality control complex. The newly-identified proteins are osteosarcoma amplified-9 (OS9), UBX domain containing-8 (UBXD8), Ubiquitinconjugating enzyme-6e (UBC6e), and ancient ubiquitous protein-i (AUP1). Each of these proteins participates in different steps of ER quality control. OS9 directs misfolded soluble glycoproteins to the dislocation complex. The mannose-6- phosphate homology domain of OS9 is involved in the recognition of glycans on the misfolded protein. UBXD8 recruits p97, the AAA+ ATPase responsible for membrane extraction of dislocated proteins, to the ER using its UBX domain. UBC6e is a membrane-anchored E2 ubiquitin conjugating enzyme. AUP1 recruits a second E2, soluble UBE2G2. Additionally, AUPI regulates substrate mono- and poly-ubiquitylation. AUPI is also necessary for lipid droplet formation. Lipid droplets are cytoplasmic organelles that store neutral lipids. Based on the data that AUPI depletion affects both ER quality control and lipid droplet formation and that pharmacological inhibition of lipid droplet formation perturbs dislocation, we propose that lipid droplet formation may also play a role in ER protein quality control.
by Elizabeth J. Klemm.
Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.
Includes bibliographical references.
The majority proteins that misfold in the endoplasmic reticulum (ER) are dislocated to the cytoplasm for degradation by the proteasome. This process of dislocation and degradation occurs in several steps. First, unfolded proteins need to be recognized in the ER. Following recognition, unfolded proteins are transported from the ER to the cytoplasm. Once in the cytoplasm, the misfolded proteins are processed and destroyed by the proteasome. This thesis examines each step of the process from the recognition of unfolded proteins in the ER, to the processing of proteins prior to degradation. We find that the murine MHC class I molecule H2-Kb expressed heterologously in yeast, is degraded in a proteasome-dependent fashion. Mere expression of H2-Kb induces the unfolded protein response. If the unfolded protein response is disrupted, degradation of H2-Kb and the misfolded, lumenal protein, T-CPY*, is greatly impaired, indicating that the unfolded protein response is essential for the recognition of unfolded proteins in the ER prior to degradation. The translocon has been implicated as the point of exit for unfolded proteins from the ER. We used existing strains that carry mutations in SEC61 that have demonstrated impaired degradation of the soluble ER protein, CPY*. We find that these strains demonstrate no defect in the degradation of the transmembrane protein HLA-A2, indicating that transmembrane and soluble proteins may interact with distinct aspects of the translocon prior to dislocation. It is hypothesized that N-linked glycans and ubiquitin must be removed from a protein prior to degradation by the proteaseome. However, we find that the disruption of PNGJ, the gene encoding yeast N-glycanase, has no effect on the degradation of CPY* or HLA-A2. To study how the removal of ubiquitin from proteins is involved in the degradation of unfolded proteins, we used a novel covalent inhibitor of de-ubiquitinating enzymes, ubiquitin-vinyl-sulfone. This inhibitor modifies 6 polypeptides in a yeast lysate, all 6 of which are known or putative de-ubiquitinating enzymes. The expression of these 6 de-ubiquitinating enzymes is not altered by heat shock, induction of the unfolded protein response or cell cycle arrest.
by Rocca Casagrande.
Ph.D.

The ER is a network of membrane sheets and tubules connected via three-way junctions. Reticulons are integral membrane proteins responsible for shaping the tubular ER. There are four reticulons isoforms in animals, two in yeast and 21 isoforms in Arabidopsis. This study developed an expression profile for isoforms AtRTNLB1-18 by expressing these reticulons driven by their native promoters. The reticulons analysed localised to the ER and coexist in many tissues. Given this result it is possible that the large size of the Arabidopsis gene family may have evolved to afford functional redundancy. It is known that a knock-out of seed specific RTNLB13 does not disrupt the seed ER morphology. Is this due to functional redundancy? In this study knock-outs were made of two other reticulons found non-exclusively in the seed, RTNLB1 and RTNLB2. In accordance with the redundancy theory, there was no apparent disruption in the mutants ER morphology. Knock-out/knock-down mutants of RTNLB1, RTNLB2 and RTNLB13 were also made and verified. The results of this were beyond the time frame of this study. In mammals and yeast reticulons are known to interact with other tubule forming proteins (DP1/Yop1p) and human atlastin. The Arabidopsis homologue of Yop1p is HVA22. In this study the seed-specific isoform HVA22b labels the tobacco ER (and the nuclear envelope), but it does not alter ER morphology or cause constrictions of the tubules (as seen with RTNLB13), suggesting that it is not a structural component. The closest plant homologue of atlastin is RHD3. This study shows that RL2, an RHD3 isoform that is highly expressed in the seed, locates to the ER without perturbing the ER morphology or Golgi body mobility. Over-expression of RL2 bearing mutations within its GTPase domain, however, induces cable-like ER, suggesting that a functional GTPase domain is required for the formation of three-way junctions. Co-expression of RTNLB13 with RL2 resulted in a striking modification of the ER network. This alteration was independent of an active RL2 GTPase domain but required a functional reticulon. RL2 and its GTPase mutants co-immunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 interact and operate synergistically in modulating ER morphology.

Soluble proteins can be targeted and inserted into the Endoplasmic Reticulum (ER) co-translationally or post-translationally, Co-translational translocation requires a ribonucleoprotein complex. Signal Recognition Particle (SRP), to target the ribosome-nascent chain complex to the ER-localised translocon. The targeting and insertion of membrane-bound proteins into the ER is more complex. One class of membrane proteins have the transmembrane domain near their C-terminus (tail-anchor proteins), and whilst the other classes of membrane proteins have had their mode of insertion elucidated, the insertion mechanism of these proteins remains unknown. This thesis deals with two areas of protein targeting in Saccharomyces cerevisiae. First the mode of insertion of Ufe1p, a tail-anchor protein, was examined by use of a variety of reporters. This work identified a region N-terminal to the transmembrane domain that is required for correct localisation of the protein. Second, the role of the SRP component, Srp72p was examined, Srp72p is essential for SRP function, but its role has not been determined. A screen was set up to identify conditional mutants of SRP72. In addition, deletions and point mutations were created to analyse the roles of conserved residues within the protein. Mutations affecting a conserved region of the protein towards the C-terminus were found to confer slight defects in SRP-dependent translocation. Intracellular localisation of the mutant proteins was identical to that of the wild-type protein. A mini-screen performed on two mutants identified multicopy suppressors of translocation defects. Finally, preliminary comparison of the binding affinities of SRP containing mutated Srp72p or wild-type SRP with the SRP receptor revealed a subtle difference.

The sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps calcium from the cytoplasm into the lumen of the endoplasmic or sarcoplasmic reticulum (ER/SR), removing excess Ca2+ from the cytoplasm and replenishing ER/SR Ca2+ stores. SERCA is located in both the ER and the ER-Golgi intermediate compartment, and so is likely maintained in the ER by retrieval. To locate the ER retrieval signal(s) in SERCA, a series of chimeric calcium pumps have been constructed. Sections of SERCA were replaced with corresponding sequence from its plasma membrane counterpart; plasma membrane calcium ATPase (PMCA). Replacing the C-terminus of SERCA with corresponding PMCA sequence results in mistargeting of the protein to the plasma membrane. The opposite construct (consisting of PMCA with the C-terminus replaced by that of SERCA) is located in the ER, suggesting that the ER retrieval signal lies towards the C-terminus of the protein. Many of the chimeras built were located in the ER. This is likely to be due to protein misfolding in some cases. Attempts were made to detect the unfolded protein response in cells expressing chimeras by measuring levels of the chaperone protein BiP. BiP upregulation was only seen when the unfolded protein response was induced pharmacologically, and not in cells expressing chimeras. More subtle mutagenesis was then carried out to assess the role of the tenth transmembrane domain of SERCA in ER retrieval and CD8 reporter constructs were used to study the tenth transmembrane domains of SERCA and PMCA. The study then focussed on determining the mechanism by which SERCA is retrieved to the ER. Rer1p and BAP31 are both candidate receptors for the retrieval of SERCA. An antibody to two epitopes in human Rer1p was raised and characterised. Immunoprecipitation and cross-linking showed that although Rer1p appears not to interact with SERCA, BAP31 shows a potential interaction and therefore could be involved in the retrieval of the calcium pump to the ER.

Mammalian/mechanistic target of mTOR complex 1 (mTORC1) regulates multiple cellular processes, including de novo protein synthesis, autophagy and apoptosis. mTORC1 overactivation occurs in a range of cancers and benign tumour dispositions as a result of mutations which increase mitogenic stimulus or cause malfunction of the tuberous sclerosis complex, the prime regulator of mTORC1 activity. mTORC1 overactivation results in elevated endoplasmic reticulum (ER) stress which, at low levels, elicits a pro-survival response. However, prolonged or excessive ER stress causes cell death. The present study utilised clinically relevant drug combinations to simultaneously enhance levels of ER stress and inhibit compensatory survival pathways in in vitro models of mTORC1 overactivity in order to cause non-genotoxic cell death. The main drugs used in this study were nelfinavir, an ER stress-inducer, chloroquine, an autophagy inhibitor, and bortezomib, a proteasome inhibitor. The key findings of this study include identification of drug combinations nelfinavir and chloroquine, nelfinavir and mefloquine, or nelfinavir and bortezomib to induce significant and selective cell death in mTORC1-driven cells, as measured by flow cytometry with DRAQ7 staining and western blot analysis for cleavage of apoptotic markers. Cell death is likely mediated through ER stress signalling, as shown by increased ER stress markers at both the level of mRNA and protein. Of interest, this study found cell death as a result of combined treatment with nelfinavir was not dependent on proteasome inhibition by nelfinavir, or autophagy inhibition by chloroquine. Additionally, nelfinavir-chloroquine-mediated cell death was completely rescued by inhibition of the vacuolar ATPase by bafilomycin-A1. In conclusion, mTORC1 overactive cells have higher basal levels of ER stress which can be manipulated with drug treatment beyond a survivable threshold, whereas cells capable of reducing mTORC1 signalling are able to survive. This study ascertained a combination of nelfinavir and chloroquine, nelfinavir and mefloquine, or nelfinavir and bortezomib, to cause effective cytotoxicity in mTORC1-driven cells.

ERp27 is a 27.7 kDa redox-inactive member of the protein disulphide isomerase (PDI) family. It was found to interact with another PDI member, the well-known thiol oxidoreductase ERp57 (58 kDa) in vitro. Although it is known that ERp57 interacts with ERp27 in vitro this interaction was not investigated in living cells. In this research project we applied in vitro and in cellulo approaches to investigate the same interaction of ERp57 and ERp27 then to compare it to the interaction of ERp57/calnexin (CNX)/calreticulin (CRT) complex to determine if the ERp57 interaction with ERp27 competes with the ERp57/CNX/CRT complex. Additionally, we investigated the physiological role of ERp27. Protein expressions and purifications were carried out by the Nickel agarose affinity chromatography to obtain sufficient amount of proteins for analysis. Additionally, proteins were purified by gel filtration-chromatography. The interaction between purified ERp27 and ERp57 was determined using isothermal titration calorimetry (ITC) and by chemical cross-linking. The ITC results confirmed the interaction between ERp57 and the lectin CRT. However, we could not detect an interaction between ERp57 and ERp27 possibly due to low protein concentrations. Moreover, the in vitro cross-linking results were in agreement with the previous research and verified the binding of ERp57 with ERp27. However, in cellulo chemical cross-linking suggested that the same interaction does not occur in living cells. Nevertheless, this investigation revealed that ERp27 binds to other proteins in cellulo. Mass spectrometry results have identified protein candidates that interact with ERp27 in living cells which are the PDI homologous P5 and the ER oxidoreductin Ero1. These results provide new insights of the role of ERp27 and provide suggestions for further research.

γ-L-glutamyl-L-cysteinylglycine known as glutathione is tripeptide synthesised in the cytoplasm. Glutathione plays important role in several cellular processes and has many functions, for example detoxification of xenobiotics, oxidative protein folding, protection against reactive oxygen species, modulation of cell proliferation and apoptosis. Glutathione is present in almost all organelles within the cell, however, it is still not known how glutathione is transported from the cytoplasm to other cellular compartments. Here we investigated the transport of glutathione into the ER where glutathione plays an important role in oxidative protein folding. Two assays to monitor glutathione transport across the ER membrane were developer and an attempt to identify the putative glutathione transporter was made. Both assays rely on selective permeability of biological membranes and use microsomes to mimic the ER environment. Microsomes were prepared from HT1080 cells expressing either redox sensitive green fluorescent protein (roGFP-iE) or glutathione S-transferase P1 (GSTP1-1A) inside the ER. By measuring the change in fluorescence of roGFP-iE it was possible to measure the rate at which glutathione is transported inside microsomes. GSTP1-1A is able to conjugate glutathione to various substrates and form a stable product, by measuring the increase in glutathione conjugates it was possible to estimate the transport of glutathione inside microsomes. Using both roGFP-iE and GSTP1-1A based assays we were able to measure glutathione transport into microsomes, both assays provide slightly different information about glutathione transport and both have different limitations. In order to identify the glutathione transporter, we used GSH as an affinity ligand and isolated all ER membrane proteins interacting with glutathione. The first approach using glutathione Sepharose beads allowed to isolate several proteins, however, all of them belonged to GST family. A second approach relied on isolating the transporter using glutathione attached to Mts-Aft-Biotin a photo activated crosslinker. Mts-Aft-Biotin approach did not result in isolation of any proteins binding specifically to GSH, this approach requires more work and needs to be improved. We showed that it is possible to isolate GSH binding proteins using GSH as affinity ligand, using this strategy it might be possible to isolate GSH transporter in the future. Both assays presented in this work are the first assays specific for the transport of glutathione across the ER membrane. In the future these assays can be used to investigate the transport of glutathione even further and contribute to the understanding of redox homeostasis in the ER.

During co-translational integration, the transmembrane (TM) sequence of a nascent membrane protein moves laterally into the ER lipid bilayer upon reaching the translocon. Our lab has previously shown that this movement is a multistep process, but it was not clear whether the observed photocrosslinking of the TM segment to translocon proteins resulted from specific interactions or simply from TM-translocon proximity. If the latter, the TM α-helix will be oriented randomly with respect to translocon proteins, whereas, if the former, a specific TM helix surface would face TRAM and/or Sec61α. Integration intermediates were prepared by in vitro translation of truncated mRNAs in the presence of a Lys-tRNA analog with a photoreactive moiety attached to the lysine side-chain. When photoadduct formation was monitored as a function of probe location within the TM α-helix, we found that the extent of photocrosslinking to TRAM and Sec61α was non-random. Thus, the TM sequence occupies a distinct location within the translocon, a result that can only be achieved through protein-protein interactions that mediate the lateral movement, positioning, and integration of the TM sequence. In the case of multi-spanning membrane proteins, it was unknown how multiple hydrophobic regions integrated into the ER membrane. By placing photoprobes within each of several TM domains of a multi-spanning membrane protein, we were able to determine at what stage of integration each TM segment was no longer adjacent to translocon proteins. Using this approach we were able to establish a mechanism of integration for multi-spanning membrane proteins co-translationally inserted into the ER membrane.

Three proteins of stripped rough microsomes (SRM) from dog pancreas or rat liver, with apparent molecular weights of 35, 56 and 90 kDa (pgp35, pp56, pp90), were phosphorylated in vitro by both ($ tau$-32P) GTP and ($ tau$-32P) ATP. Another SRM protein of 15 kDa (pp15) was phosphorylated in vitro only by ($ tau$-32P) GTP. Comparison of the in vitro phosphorylation profile of SRM to those of other well defined subcellular fractions, i.e., plasma membranes, Golgi apparatus, lysosomes, endosomes, mitochondria and smooth microsomes, showed the four major GTP phosphorylated proteins were restricted to the endoplasmic reticulum (ER). Further characterization of these proteins showed them to be phosphorylated on serine residues at the cis face of SRM and to be integral membrane proteins. The 35 kDa protein was glycosylated, with 2 N-linked oligosaccharide sidechains, therefore possessing a luminal domain. Competition and inhibition studies showed that GTP and ATP phosphorylation of the proteins were distinct. Regulation of GTP specific phosphorylation was by adenosine nucleoside. Purification of the 35 and 90 kDa phosphoproteins was achieved, along with another nonphosphorylated glycoprotein of 25 kDa, as a phosphoglycoprotein complex which elutes from Sephacryl S300 chromatography at an apparent molecular weight of about 400,000. Partial Sequence Receptor (Weidmann et al, (1987) Nature 328: 830-833), suggesting that this phosphoglycoprotein complex may form part of the translocation apparatus of the rough ER. These results suggest a role for phosphorylation of ER membrane proteins in the process of translocation.

Isolated rough and smooth microsomes of the endoplasmic reticulum and isolated Golgi apparatus from rat liver were analyzed by proteomics using mass spectrometry, identifying 1064 proteins among the three fractions. An additional 598 proteins were identified by biochemically subfractionating the rough and smooth microsomes by treatment with a high salt wash and the detergent Triton-X 114. Proteins were quantified by redundant peptide counts which enabled an assessment of the extent of cross-contamination between the endoplasmic reticulum and Golgi fractions and other organellar contamination. Results of this analysis revealed that the Golgi fraction was contaminated up to 30% by proteins of the endoplasmic reticulum and that the mitochondria constitute the largest source of organellar contamination for all three fractions. Hierarchical clustering of the distribution profiles of proteins among the three fractions assigned proteins to either the rough and/or smooth endoplasmic reticulum or the Golgi apparatus. In doing so, the protein disulphide isomerase, ERp44, was localized to the Golgi. This result was verified by immunolocalization with an ERp44 antibody. Furthermore, hierarchical clustering assigned a location for 176 previously uncharacterized proteins in the endoplasmic reticulum providing a subcellular context to their putative functions predicted by bioinformatics. Additionally, the biochemical subfractionation of the rough and smooth microsomes assigned proteins to the cytosolic, membrane or luminal subcompartments of the endoplasmic reticulum. These results guided the selection of uncharacterized membrane proteins for further characterization leading to the identification of 7 proteins upregulated by ER stress, which included 4 new molecular chaperones. Finally, a comparison of this work with previous proteomics analyses of these organelles showed that the proteomes of the endoplasmic reticulum and Golgi apparatus presented here may be the mo
L'analyse par protéomique des microsomes du Réticulum Endoplasmique rugueux, des microsomes du Réticulum Endoplasmique lisse et de l'appareil de Golgi a permis l'identification de 1064 protéines par spectrométrie de masse. Par ailleurs, le fractionnement biochimique des microsomes lisses et rugueux par lavage avec une solution saline concentrée suivi d'un traitement au détergent Triton X-114 a permis l'identification de 598 nouvelles protéines. Les protéines furent quantifiées en fonction du nombre de peptides identifiés par spectrométrie de masse. La quantification des protéines a permis d'évaluer le degré de contamination croisée présent dans les fractions du Réticulum Endoplasmique, de l'appareil de Golgi et celui provenant des autres organelles. Les résultats de cette analyse ont révélé que la fraction de Golgi était contaminée jusqu'à un maximum de 20% par les protéines provenant du Réticulum Endoplasmique et que les mitochondries constituaient la source essentielle de contamination dans ces trois fractions. La clustérisation hiérarchique des protéines quantifiées a permis de dresser le profile de distribution des différentes protéines et ainsi de les assigner au sein des différents compartiments, à savoir aux microsomes du Réticulum Endoplasmique rugueux et/ou lisses, ou alors à l'appareil de Golgi. De ce fait, la protéine disulphide isomerase, ERp44, a été localisée dans l'appareil de Golgi. Ce résultat a été confirmé par immunolocalisation avec l'anticorps ERp44. Par ailleurs, cette même clustérisation hiérarchique a permis de localiser pour la première fois 176 protéines dans le Réticulum Endoplasmique correspondant ainsi à leur fonction putative prédite par bioinformatique. De plus, le fractionnement biochimique des microsomes lisses et rugueux a permis d'assigner les protéines dans les compartiments subcellulaires du Réticulum Endoplasmique : cytosol, membrane ou lumière. Ces résultats ont é

Adipose tissue plays a central role in the regulation of metabolic homeostasis. In obesity adipocytes are challenged by many insults: surplus energy, inflammation, insulin resistance and considerable endoplasmic reticulum (ER) stress. ER stress has been casually linked to increased inflammation and insulin resistance. Also, obesity linked type 2 diabetes is associated with hyperglycaemia, lipotoxicity and endotoxemia. Therefore, the aims of this thesis briefly were to 1) characterise human pre-adipocytes during differentiation, as a suitable primary cellular model to examine intracellular pathways, 2) investigate the role of glucose and fatty acids on ER stress pathway; as these primary insults are considered to have clear impact on inflammation, insulin resistance (IR) status and diabetes pathogenesis 3) to examine the role of lipopolysaccharide (LPS), a gut derived bacterial fragment, on ER stress; as LPS is now considered a systemic circulating factor raised in conditions of IR, 4) the role of salicylate, known to have anti-inflammatory properties which may negate or at least attenuate the effects of ER stress. Components of the ER stress pathways were studied in human abdominal subcutaneous (AbSc) adipose tissue (AT) from obese and lean subjects. Following characterisation, culture and differentiation of primary human pre-adipocytes, these adipocytes were treated with lipopolysaccharide (LPS), high glucose (HG), tunicamycin (Tun) and saturated fatty acids (SFA) either alone or in combination with sodium salicylate (Sal). Quantitative RT-PCR, western blotting, adipokine analysis were used to assess expression levels. Markers of ER stress were significantly increased in AbSc AT from subjects with obesity (P<0.001). Differentiated primary human adipocytes treated with LPS, Tun, HG and SFA showed significant activation of p-eIF2α and ATF6 and their downstream targets (P<0.05). This effect was alleviated in the presence of Sal. There was also significant activation of AktSer473 during ER stress (P<0.05). This thesis presents important evidence that firstly, there is increased ER stress in human adipose tissue of obese individuals, secondly, LPS, hyperglycaemia and saturated fatty acids induce significant ER stress in primary human adipocytes and finally that induction is alleviated by salicylate. Taken together these studies highlights that ER stress occurs in human differentiated pre-adipocytes is exacerbated in conditions of high glucose, high saturated fatty acids and LPS, as well as determining that such primary insults can be reduced by salicylates providing initial evidence that therapeutic agents have the potential capacity to alleviate ER stress in human adipose tissue.

The functional integrity of the proteome is essential for proper cell functioning. Protein homeostasis, or proteostasis, is maintained by a network of pathways that mediate the biosynthesis, folding and degradation of proteins. Accumulating evidence suggest that ageing is associated with a general decline in protein homeostasis. Proteins are synthesised in the cytosol as extended polypeptide chains, which must then be folded in to their native conformation, before moving to their site of function. Protein folding occurs in different subcellular compartments, such as the cytosol and the endoplasmic reticulum (ER). Due to the nature of the proteins that are processed in the ER (e.g. ionic channels, receptors, hormones, signalling molecules), the function of this compartment can be seen to be of vital importance to the cells. However, the effect of age on ER protein homeostasis is virtually unknown. A combination of post-mortem mouse tissues and cell-based models were used to examine the impact of age on ER protein folding, quality control and the ER stress response. Analysis of the expression level of ER-resident and ER-linked proteins showed a number of age-related changes in mouse tissues, in human fibroblasts aged in vitro by serial passage, and human fibroblasts obtained from young and old donors (in vivo aged cells). Overall, the pattern of changes was variable between different tissues and cell systems. However, a common feature of aged tissues and both cellular models of ageing, was a significant increase in phosphorylation of eIF2alpha, indicating that ER protein homeostasis is affected with age. In addition, ageing in several tissues and in both cellular systems was associated with accumulation of polyubiquitinated substrates, suggesting that degradation of abnormal proteins via the proteasome is deficient. More detailed investigation of ER proteostasis using the cellular models showed that in vitro aged cells had a decreased capacity to fold a temperature sensitive model membrane protein (ts O45 VGV-G) and were less efficient at degrading two model substrates of the ER-associated degradation pathway. Since the ER protein folding and degradation pathways are modulated by ER stress response signalling to restore ER homeostasis, therefore their malfunctioning would impact on the ability of aged cells to cope with stress. Indeed, in the aged cells both PERK and IRE-1 signalling were perturbed. In addition, the level of BiP was not upregulated following ER stress in the aged cells. Moreover, ER stress induction led to decreased cell survival in the case of aged cells, showing that the inability of aged cells to maintain ER protein homeostasis results in increased susceptibility to cell death. All these data together shows how perturbed ER proteostasis can occur with increased age and its impact on overall cell wellbeing, therefore provide new insight into mechanisms of cell ageing.

Selenium (Se) is associated with insulin resistance and may affect endothelial function thereby increasing the risk of type 2 diabetes and associated cardiovascular disease (CVD). However, the molecular mechanisms involved are not clear. The endoplasmic-reticulum (ER) stress response is a mechanism involved in apoptosis induced by high Se in some cancer cells and, also in the pathogenesis of insulin resistance and endothelial dysfunction (ED). Thus, we hypothesised that high Se status causes ED through ER stress response. Endothelial cells (HUVECs) and EA.hy926 cell lines were treated with selenite (0.5-10 µM) for 24 hours in the presence or absence of the ER chemical chaperone, 4-phenylbutryic acid (PBA). ER stress markers were investigated using qPCR and western-blot technique. Endothelial function was assessed by the Griess assay, flow cytometry, Matrigel® and colourimetric assays. Data were expressed as S.E.M (p < 0.05) vs. control. High Se concentration (5-10 µM) compared to physiological concentration (0.5–2.0 µM) enhanced mRNA expression of ER-stress markers:- activating transcription factor-4 (ATF4), CAAA/enhanced-binding homologous protein (CHOP) and X-binding box-1 (XBP-1). In addition, high selenite concentration reduced nitric oxide production and angiogenic capacity in endothelial cells. Moreover, high selenite treatment significantly (p < 0.05) increased production of reactive oxygen species (ROS) and induced apoptosis through caspase-3/7 activity. Interestingly, PBA completely reversed all the effects of high selenite on endothelial function, indicating the involvement of the ER-stress response. High Se treatment caused endothelial dysfunction through the activation of the ER-stress response. This thesis additionally warns the public to be aware of the risks of the use of Se supplements as a prophylactic agent against oxidative-stress disease.

Investigators have been attempting to identify the receptor for ribosomes on the rough endoplasmic reticulum (RER) for almost 20 years, yet the ribosome receptor has remained elusive. Rough microsomal membranes contain endogenous ribosomes bound in at least two types of interactions. Loosely associated ribosomes can be removed by extraction with a high ionic strength solution, but ribosomes that were actively engaged in translocation when the membranes were isolated remain tethered to the membrane by a nascent polypeptide (Adelman et al., 1973). The original assay for the ribosome receptor involved stripping all of the endogenous ribosomes off of intact membranes before adding back a quantitated amount of ribosomes. More recent assays have employed detergent solubilization of the membrane and then reconstitution of the membrane proteins into lipid vesicles before adding back ribosomes. In both cases ribosome binding to its receptor is measured in an assay that does not involve translation or translocation. We utilized a crosslinking assay to attempt to identify membrane proteins that function as a binding site for ribosomes engaged in protein translocation across the endoplasmic reticulum. In vivo bound ribosomes that remain associated with the membrane after extraction with a high ionic strength solution are likely to be bound to a functional translocation site. The water soluble, membrane impermeable, thiol-cleavable crosslinker 3,3'-dithiobis (sulfosuccinimidylpropionate) was selected to limit reaction to protein domains located on the cytoplasmic face of salt extracted microsomal membrane vesicles. A specific subset of RER proteins was reproducibly crosslinked to the endogenous ribosomes. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35 kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180 kD protein that had recently been proposed to be a ribosome receptor (Savitz and Meyer, 1990). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes in an in vitro binding assay to determine whether ribosome binding activity could be ascribed to the 180 kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180 kD protein, as determined by Coomassie blue staining of a polyacrylamide gel. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments and functional assays with proteoliposomes demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180 kD protein by chromatography. Taken together, the evidence indicates that the 180 kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor. To continue the investigation of ribosome binding, an assay was designed to characterize ribosome-nascent chain complexes bound to the microsomal membrane during translocation. A series of translocation intermediates consisting of discrete sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. Proteinase K was then used as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport. Only those partially translocated nascent chains of 100 amino acids or less were insensitive to protease digestion by externally added protease. It was concluded that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, shorter nascent polypeptides appear not to have lumenally exposed segments. Ultimately, a functional assay for the ribosome receptor should include binding studies conducted under physiological conditions. For this purpose, an assay was developed that allowed translation, translocation, and termination of a secretory protein to be monitored with probes designed to independently quantitate translating and non-translating ribosomes. A synchronized wheat-germ translation system was programmed with bovine preprolactin mRNA and aliquots were taken at various time points before and after adding membranes. The samples were then separated into membrane bound and soluble species by centrifugation. RNA was isolated from each supernatant and pellet sample and blotted onto nylon sheets. By probing the dot blots with probes that hybridize with either the 5S RNA of wheat germ ribosomes or the preprolactin transcript, the translating ribosomes could be monitored without the interference of the endogenous canine ribosomes on the membrane. By comparing the total amount of preprolactin transcript that bound to the membrane versus the total amount of wheat germ ribosomes bound to the membrane, it was discovered that the vast majority (>99%) of wheat germ ribosomes that bound to the microsomal membrane were non-translating ribosomes. In later experiments it was found that the non-translating ribosomes did not compete with the translating ribosomes; under all conditions tested, the translating ribosomes had access to translocation sites on the microsomal membrane. One interpretation of this data is that all ribosome binding sites are not identical. It may be that functional sites for translocation are a distinct subclass of total ribosome binding sites. Another interpretation is that a ribosome in a nascent chain-SRP complex has a much higher affinity for the ribosome receptor than nontranslating ribosomes or 60S subunits. Perhaps the non-translating ribosome can not compete with ribosomes engaged in translocation. As stated earlier, ribosomes do make at least two kinds of interactions with the microsomal membrane surface. This data may be indicative of those types of interactions.

Obesity is the most significant risk factor for developing type II diabetes mellitus (T2DM). Obesity induces adipocyte endoplasmic reticulum (ER) stress, prior to onset of insulin resistance. A pathological inability of white adipose tissue (WAT) to expand to accommodate excess energy is predominantly due to impaired adipogenesis. The research hypothesis was that ER stress in human WAT is important in inducing WAT dysfunction and subsequent insulin resistance and T2DM. The aims of this study were to elucidate interactions of ER stress in human WAT and to characterise the role of ER stress in human adipogenesis. Abdominal SAT biopsies and anthropometry were collected from T2DM subjects before and after bariatric surgery and non-diabetic subjects. Preadipocytes were extracted from human WAT and differentiated into adipocytes. Lipogenesis, lipolysis, glucose uptake, insulin sensitivity, and ER stress and adipogenesis gene and protein expression were assessed in control cells and with ER stress inducers, inhibitors or siRNA. The results of this study found both restrictive and malabsorptive bariatric interventions are effective weight loss interventions for obese T2DM patients and result in significantly improved glucose and insulin levels six months after surgery. WAT health is better following restrictive procedures as shown by lower and better regulation of ER stress markers. Adipogenesis in primary human preadipocytes is influenced by adiposity and WAT depot and the IRE1-XBP1s UPR is essential in human adipogenesis. XBP1s plays a vital role upstream of CEBP and PPAR in human adipogenesis and it is necessary for mediating the action of insulin. Wnt10b plays an inhibitory role in human adipogenesis and acts independently of XBP1s. Collectively these findings suggest that WAT function is key for metabolic health and can be impaired by ER stress; however regulated adipogenesis may serve to improve WAT function and therefore improve metabolic health.

Vascular calcification (VC) is a health problem common in ageing populations, diabetes and chronic kidney disease. It leads to vascular stiffening and heart failure. VC is a regulated process mediated by vascular smooth muscle cells (VSMCs), with similarities to developmental osteogenesis. The exact molecular events responsible for triggering it are unknown. The endoplasmic reticulum (ER) is involved in folding of proteins. ER stress occurs as a result of unfolded protein accumulation and has been implicated in osteoblast differentiation and bone mineralization. Therefore, I hypothesized that ER stress signalling regulates osteogenic differentiation and calcification of VSMCs. I showed that calcification of human aortas was associated with changes in ER stress marker expression. Warfarin and TNFα, which are both established inducers of vascular calcification, increased expression of ER stress markers in VSMCs. ER stress modelled in human primary VSMCs in vitro increased their calcification and was shown to modulate expression of a number of bone related genes, such as BMP-2, Runx2, Osterix, ALP, BSP and OPG in VSMCs in vitro. I also demonstrated that ER stress activated features characteristic of a secretory phenotype in VSMCs, such as downregulation of SMC markers and components of TGFβ signalling related to contractile differentiation, as well as BMP-2. Taken together these results suggested that ER stress can induce changes that lead to osteogenic differentiation. To further explore the relationship between ER stress and osteogenic differentiation of VSMCs Osterix and ALP were studied in more detail. ALP activity was upregulated by ER stress, but did not change when VSMCs calcified. Promoter analysis showed that ALP might be regulated by ER stress via indirect mechanisms and potential regulators of ALP transcription were identified using proteomic analysis.

Alzheimer’s Disease (AD) is the most frequent form of dementia. A small percentage of cases is inherited (Familial AD, FAD) and is due to dominant mutations on three genes, coding for Amyloid Precursor Protein (APP), Presenilin-1 (PS1) and Presenilin-2 (PS2). Mutations in these proteins cause alterations in the cleavage of APP by a PS1- or PS2- containing enzyme, named У-secretase, thus leading to an increase in the ratio between Aß42 and Aß40, the two main peptides finally derived from APP maturation. This in turn would increase the deposition of the “Amyloid Plaques”, one of the main histopathological feature of AD. To date, the generation of Aß42 peptides, its oligomers and finally amyloid plaques is the core of the most widely accepted pathogenic hypothesis for AD, the “Amyloid Cascade Hypothesis”. PS1 and PS2 are ubiquitous “9 trans-membrane domains” homologous proteins localized mainly in the membranes of Endoplasmic Reticulum (ER), Golgi apparatus, endosomes and plasma membrane. Despite being the catalytic core of У-secretase, PSs display also some specialized, У-secretase independent activities. On this line, numerous studies reported a role for FAD-linked PS mutations in cellular calcium (Ca2+) alterations. Ca2+ is a key second messenger in living cells and it regulates a multitude of cell functions; thus, alterations in its signaling cascade can be detrimental for cell fate. Ca2+ mishandling has been proposed as a causative mechanism for different neurodegenerative diseases and in particular for AD. Although supported by several groups for many years, the Ca2+ hypothesis for AD pathogenesis has never been undisputedly accepted, since some data were clearly in contrast, especially those considering PS2 mutations. In our lab, it was previously shown that several FAD PS2 mutants, but not PS1, reduce ER and Golgi apparatus Ca2+ content, mainly by interfering with SERCA activity. Over the last decade, evidence has accumulated on the existence of continuous flux of information between the ER and mitochondria, two organelles whose privileged interplay modulate key aspects of cell pathophysiology, ranging from lipid metabolism and Ca2+ homeostasis to cell death. Several proteins have been suggested to be involved in keeping the ER and mitochondria at a given distance, allowing the correct organization, their mutual interactions and Ca2+ cross-talk. Among them, mitofusin 2 (Mfn2), which is located on both the outer mitochondrial membrane (OMM) and the ER surface, has been shown to take part in homotypic interactions that contribute to the tethering at the level of mitochondria-associated-membranes.(MAMs). Interestingly, also PS1 and PS2 are enriched in MAMs: we have recently demonstrated that PS2, but not PS1, is able to modulate ER-mitochondria tethering and their Ca2+ cross-talk, with PS2-FAD mutants more potent than their wt counterpart in this novel function. We here investigate the molecular mechanism by which PS2 favours ER-mt tethering, taking into consideration the possibility that PS2 effect depends on the presence of Mfn2. By crossed genetic complementation and ablation experiments, we found that, in order to modulate ER-mitochondria coupling, PS2 requires the expression of Mfn2 and viceversa. In contrast, their homologues PS1 and Mitofusin 1 (Mfn1) are completely dispensable for these functions. Functional and biochemical evidence indicates that PS2 (wt and FAD) needs to physically interact, via its big cytosolic loop, with Mfn2 at both sides of MAM domains, likely forming, or stabilizing, a triple complex made by itself, ER and mitochondrial Mfn2. Our results clearly suggest that PS2 and Mfn2 cooperate and need one each other to promote ER-mitochondria apposition. On the contrary, their homologues PS1 and Mfn1 are completely dispensable in this function. In order to explain the stronger effect of FAD-PS2 compared to wt, we performed protein subcellular fractionation from mouse brains, and we observed that in transgenic (tg) mice, carrying FAD-PS2-N141I mutation, PS2 is strongly enriched in MAMs, compared to controls. Moreover, in tg mice also Mfn2 levels are slightly increased in MAMs, thus possibly explaining the stronger tethering in presence of FAD mutations. We proposed a model in which, being FAD-PS2 more enriched in MAMs compared to wt, it could here recruit more Mfn2 (by physically interacting with it both in cis and in trans) and form more PS2-Mfn2 complexes critical for determining the apposition between the two organelles. Finally, the increase in ER-mitochondria coupling was observed not only in FAD-models over-expressing the mutated form of PS2, but also in human fibroblasts from patient carrying the PS2-N141I mutation, thus a condition in which the mutated protein exerts its function independently of any artefact due to its over-expression. Further investigations will be focused to highlight the mechanism that promotes accumulation into MAMs of FAD-PS2 and whether these stronger FAD-PS2-linked effects on ER-mitochondria coupling are involved in the pathogenesis of AD
La malattia di Alzheimer (AD) è la forma più frequente di demenza. Una piccola percentuale dei casi è ereditaria (Familial AD, FAD) ed è dovuta a mutazioni autosomiche dominanti in tre geni, che codificano per la Proteina Precursore dell’Amiloide (APP), per Presenilina-1 (PS1) e per Presenilina-2 (PS2). Le mutazioni in queste proteine causano alterazioni nella maturazione di APP da parte di un enzima (detto У-secretasi) che contiene alternativamente PS1 o PS2, portando così ad un aumento del rapporto tra Aß42 e Aß40, che sono i due principali peptidi prodotti in seguito al taglio di APP. Questo a sua volta induce un aumento della deposizione delle “placche amiloidi”, uno dei principali marcatori isto-patologici dell’AD. La generazione del peptide Aß42, dei suoi oligomeri e delle placche amiloidi costituisce il core dell’ipotesi patogenica ad oggi più accreditata per spiegare l’insorgenza della malattia di Alzheimer, ossia “l’ipotesi della cascata amiloide”. PS1 e PS2 sono proteine omologhe ubiquitarie con 9 domini trans-membrana, principalmente localizzate nelle membrane del reticolo endoplasmatico (ER), dell’apparato del Golgi, degli endosomi e in membrana plasmatica. Oltre a costituire il nucleo catalitico della У-secretasi, le preseniline possiedono anche delle attività specializzate У-secretasi indipendenti, talvolta non ridondanti tra le due proteine. Per esempio, molti studi hanno evidenziato un ruolo per alcune mutazioni nelle Preseniline associate a FAD nell’alterazione dell’omeostasi del calcio (Ca2+) intracellulare. Il Ca2+ è un secondo messaggero chiave per le cellule e regola numerose funzioni cellulari; pertanto, alterazioni nelle dinamiche di questo ione possono essere estremamente dannose per le cellule e sono state proposte essere alla base di diverse patologie neurodegenerative, tra cui in particolare l’AD. Sebbene sia stata ampiamente supportata da vari gruppi, l’ipotesi dell’alterazione dell’omeostasi del Ca2+ alla base dell’insorgenza dell’AD non è mai stata completamente accettata, dal momento che alcuni dati sono chiaramente discordanti, soprattutto per quanto riguarda alcune mutazioni in PS2. Nel nostro laboratorio, è stato in precedenza dimostrato che diverse mutazioni associate a FAD in PS2, ma non in PS1, riducono il contenuto di Ca2+ nell’ER e nell’apparato del Golgi, principalmente attraverso una inibizione della pompa SERCA. Negli ultimi anni, crescenti evidenze hanno dimostrato l’esistenza di un continuo flusso di informazioni tra l’ER e i mitocondri, due organelli la cui interrelazione modula aspetti chiave nella pato-fisiologia delle cellule, dal metabolismo lipidico alla regolazione dell’omeostasi del Ca2+, fino alla morte cellulare. Diverse proteine sono state coinvolte nel mantenimento di una appropriata distanza tra l’ER e i mitocondri, permettendo così una corretta organizzazione delle loro reciproche interazioni e dello scambio di Ca2+ tra di essi. Tra queste è stato proposto che Mitofusina-2 (Mfn2), localizzata sia sulla membrana mitocondriale esterna (OMM) che, in minore percentuale, sulla superficie dell’ER, moduli il tethering fisico tra i due organelli, attraverso delle interazioni di tipo omotipico a livello delle “membrane associate ai mitocondri” (MAM). Anche PS1 e PS2 sono arricchite a livello delle MAM: nel nostro laboratorio abbiamo recentemente dimostrato che PS2, ma non PS1, modula la vicinanza fisica tra l’ER e i mitocondri, influenzando così lo scambio di Ca2+ tra di essi. Nel lavoro presentato in questa tesi, abbiamo studiato attraverso quale meccanismo molecolare PS2 è in grado di influenzare il tethering, prendendo in considerazione la possibilità che l’effetto di PS2 dipenda in qualche modo dalla presenza di Mfn2. Attraverso esperimenti incrociati di ablazione e ricostituzione genetica, abbiamo dimostrato che per modulare l’accoppiamento tra i due organelli PS2 necessita dell’espressione di Mfn2, e viceversa. Al contrario, le loro proteine omologhe PS1 e Mitofusina-1 (Mfn1) non sembrano essere coinvolte in queste funzioni. Evidenze funzionali e biochimiche indicano che PS2 (wt e FAD) interagisce fisicamente attraverso il suo esteso dominio citosolico con Mfn2 presente in entrambi i lati delle MAM, probabilmente formando o stabilizzando un triplice complesso formato da PS2, da Mfn2 sull’ER e da Mfn2 sulla OMM. I risultati qui esposti suggeriscono che PS2 e Mfn2 cooperano e necessitano reciprocamente una dell’altra per mantenere il tethering tra ER e mitocondri, mentre invece PS1 e Mfn1 non sono necessarie. Per spiegare l’effetto potenziato sull’accoppiamento tra i due organelli delle forme mutate di PS2 associate a FAD, rispetto alla proteina wt, abbiamo effettuato dei sub-frazionamenti proteici a partire da cervelli di topi. Ciò che abbiamo osservato è che nei topi transgenici (tg), che portano la mutazione associata a FAD PS2-N141I, PS2 è fortemente arricchita a livello delle MAM, rispetto ai controlli. Inoltre, nei topi transgenici anche Mfn2 è leggermente arricchita nelle MAM, il che potrebbe forse spiegare l’effetto più forte sul tethering delle mutazioni FAD. Il modello che proponiamo è che, essendo la PS2 mutata più arricchita nelle MAM rispetto alla forma wt, recluti più Mfn2 (interagendo con essa sia in cis che in trans) e formi più complessi PS2-Mfn2, i quali sono fondamentali per determinare l’accoppiamento tra i due organelli. Infine, l’aumentato tethering tra ER e mitocondri è stato osservato anche in fibroblasti di un paziente FAD con la mutazione PS2-N141I, ossia una condizione in cui la proteina mutata esercita la sua funzione indipendentemente da qualsiasi possibile artefatto legato alla sua sovra-espressione. Ulteriori studi saranno necessari per capire quale meccanismo favorisce l’accumulo di PS2 mutata nelle MAM e se l’aumentata vicinanza tra ER e mitocondri, osservata in presenza delle mutazioni in PS2 associate a FAD, sia in qualche modo coinvolta nell’insorgenza o, quantomeno, nella progressione della malattia di Alzheimer

Beta-cell failure is a key step in the progression from metabolic disorder to overt type 2 diabetes (T2D). This failure is characterised by both secretory defects and loss of beta-cell mass, the latter most likely through increases in the rate of apoptosis. Although the mechanisms underlying these beta-cell defects are unclear, evidence suggests that chronic exposure of beta-cells to elevated fatty acid (FA) plays a role in disease development in genetically susceptible individuals. Furthermore, it has been postulated that endoplasmic reticulum (ER) stress signalling pathways (the unfolded protein response; UPR) play a role in FA-induced beta-cell dysfunction. The broad aim of this thesis was to explore the nature of these relationships. Experiments detailed in this thesis demonstrate that MIN6 beta-cells mount a comprehensive ER stress response with exposure to elevated saturated fatty acid palmitate, but not the unsaturated fatty acid, oleate, within the low elevated physiological range. This response was time-dependent and involved both transcriptional and translational changes in UPR transducers and targets. The differential activation of ER stress in MIN6 beta-cells by saturated, but not unsaturated FA species may represent a mechanism of differential beta-cell death described in many studies with these FA. Furthermore, these experiments describe defects in ER to Golgi trafficking with chronic palmitate treatment, but not oleate or thapsigagin treatment, identifying this as a potential mechanism by which palmitate treatment induces ER stress. Moreover, these studies have shown the relevance to ER stress to a whole body model of T2D by demonstrating UPR activation in the islets of the db/db mouse. In conclusion, studies detailed in this thesis have demonstrated that ER stress occurs in in vitro and in vivo models of beta-cell lipotoxicity and apoptosis. In addition, these studies have identified defects in ER to Golgi trafficking as a mechanism by which palmitate treatment induces ER stress. These studies highlight the importance of ER stress in the development of T2D.

The ubiquitin ligase gp78, involved in ER-associated degradation, has been extensively studied using the 3F3A monoclonal antibody, and correlates with increased metastasis incidence in cancer patients (review in Chiu et al. 2008) The 3F3A-labeled ER characterized as an ER domain in close association with mitochondria distinct of the reticulon-labeled ER tubules, the juxtanuclear and the perinuclear ER. Overexpression of gp78 saw 3F3A expansion to the peripheral ER, but remained in association with mitochondria. This association is calcium-sensitive as elevation in cytosolic calcium levels disrupted contact with mitochondria. Similarly, induction of calcium released from the ER through thapsigargin or ATP stimulation of purinegic receptors promoted dissociation of 3F3A labeled ER and mitochondria. Upon ER-mitochondria dissociation, the IP3R-labeled ER dissociated in a distinct domain from the 3F3A-labeled ER, indicating that regulation of the ER-mitochondria association by free cytosolic calcium is a characteristic of smooth ER domains and that multiple mechanisms regulate the interactions between these organelles. Upon overexpression of gp78, the 3F3A labeled peripheral ER was identified as the site of gp78-mediated ubiquitylation. Gp78 polyubiquitylated substrates where stabilized through Cue domain interaction in the peripheral ER. Derlin-1 and derlin-2, involved in retrotranslocation of ERAD substrates, localized to a juxtanuclear ER domain, where ubiquitylated proteins accumulate upon proteasome inhibition. This segregation was shown in a non-overexpressing system using HT-1080 fibrosarcoma cells expressing elevated levels of endogenous gp78. This shows the special segraqgation of ERAD by gp78-mediatedubiquitin ligase activity in the peripheral ER. This may function in ER quality control and regulate the expression of smooth ER-associated substrates in response to physiological change. Finally, we found that stimulation of gp78 at the cell surface by its ligand AMF disrupts ER-mitochondria interaction, calcium coupling and decreases gp78 ubiquitin ligase activity. Disruption of the ubiquitin ligase activity via mutation of gp78 RING domain or expression of ubiquitin mutant unable to form elongated polyubiquitin chains disrupts ER-mitochondria coupling. The increased fragmentation and reduced mobility of mitochondria upon gp78 overexpression were dependant on ubiquitin ligase activity and prevented by AMF treatment. These results describe a novel mechanism regulating mitochondrial dynamics via the ER ubiquitin ligase gp78.

Apoptosis is a genetically programmed highly regulated form of cellular suicide that plays an essential role in the development and tissue homeostasis of multicellular organisms. As a variety of pathological states such as cancer, autoimmune and neurodegenerative diseases can be ascribed to the deregulation of the apoptotic program, understanding the molecular mechanisms underlying this process is a necessary first step to therapeutic intervention. The BCL-2 family of proteins is of paramount importance in the regulation of apoptosis through their control of caspases, a family of cysteine proteases responsible for cellular demolition. The p53 tumour suppressor protein is a transcription factor that eliminates potentially dangerous cells via activation of the apoptotic program through the regulation of various genes, including those of the BCL-2 family. I found that BIK, a member of the pro-apoptotic BH3-only class of BCL-2 homologues, is upregulated by p53. Unlike all other BH3-only proteins however, BIK was found to be uniquely localized to membranes of the endoplasmic reticulum. BIK was induced in response to several stress stimuli, including genotoxic stress (radiation; doxorubicin) and over-expression of E1A or p53, but not by ER stress pathways resulting from protein misfolding. Using siRNA technology, I showed that BIK plays a critical role in p53-induced cell death by acting at the ER to trigger Ca2+ release, mitochondrial fission, BAX/BAK activation, cytochrome c release, caspase activation and apoptosis. BIK also stimulated the (BCL-2 inhibited) accumulation and oligomerization of BAK at ER membranes. Cells doubly deficient of both BAX and BAK were resistant to ER Ca2+ release and apoptosis by ectopic expression of both BIK and p20BAP31, suggesting that these multidomain pro-apoptotic BCL-2 proteins may serve as a common checkpoint at the ER for varying modes of stress stimuli. Thus, p53 appears to employ BIK as part of its apopto

The Bcl2 family proteins are central regulators of apoptosis; the primary form of physiological cell death. Furthermore, inappropriate activation or silencing of Bcl2 family members has been associated with cancer development and treatment resistance. These proteins therefore represent attractive therapeutic targets, and, in consequence, an increased understanding of the roles played by Bcl2 proteins will be important for the development and implementation of targeted therapies. Although best characterized with respect to their role at the mitochondria, it is now evident that Bcl2 proteins also function at the endoplasmic reticulum (ER). The focus of this thesis is on the role of Bcl2 proteins at the ER, specifically with respect to cell death initiated by the ER localized proteins Bik and p20Bap31. Bik and p20Bap31 were previously shown to initiate similar proapoptotic pathways, characterized by an early release of ER calcium stores, and inhibitable by Bcl2. The results of the current study are two-fold: first, using ER-targeted Bak and Bcl2 (Bakb5 and Bcl2b5) in a Bak/Bax deficient background, I have shown that Bik can disrupt an interaction between Bak and Bcl2 at the ER. Furthermore, Bik could overcome the protective effect of Bcl2b5 with respect to Bakb5. This finding provides the first direct evidence for regulation of cell death via binary interactions between Bcl2 family members at the ER. The second part of this study was designed to determine the roles of Bax/Bak and ER-restricted Bcl2 in the p20Bap31-initiated pathway. Using E1A/DNp53 transformed wild-type and Bax/Bak double knockout baby mouse kidney epithelial cells, I have shown that ectopic expression of p20Bap31, but not of Bik, can initiate a paraptosis-like form of non-apoptotic, Bax/Bak independent, cell death. Of particular importance, cell death could be delayed by Bcl2b5, in the absence of Bax/Bak; pointing to a novel, Bax/Bak independent, prosurvival role for Bcl2 at the ER. In summary, this study demonstrates the ability of Bcl2 family members to regulate cell death through binary interactions at the ER, and of Bcl2 to inhibit cell death in a Bax/Bak independent manner. Furthermore, a novel non-apoptotic cell death pathway initiated by p20Bap31 expression is identified.
La famille de protéines Bcl2 a une importance fondamentale dans le contrôle de l'apoptose; la forme principale de mort cellulaire physiologique. De plus, le développement de cancers et la résistance aux thérapies sont associés à l'activation ou la suppression de membres de la famille Bcl2. Cette famille de protéines représente une cible thérapeutique intéressante et par conséquent, une connaissance approfondie des rôles des protéines Bcl2 sera important pour amener et exécuter des thérapies ciblées. Tandis que le rôle de ces protéines à la mitochondrie est bien caractérisé, une fonction des protéines Bcl2 au niveau du réticulum endoplasmique (RE) est maintenant évidente. Cette thèse porte sur le rôle des protéines Bcl2 au niveau du RE, particulièrement concernant l'apoptose initiée par Bik et p20Bap31, deux protéines qui sont localisés au RE. Les voies de signalisation proapoptotique qui sont initiées par Bik et p20Bap31 sont caractérisées par une libération précoce d'une réserve de calcium du RE et sont inhibées par Bcl2. Les résultats de l'étude présente portent sur deux domaines: premièrement, en empruntant des formes de Bak et Bcl2 qui sont ciblées au RE (Bakb5 et Bcl2b5) sur un fond déficient en Bak et Bax, je démontre que Bik est capable de perturber l'interaction entre Bak et Bcl2 au RE. En outre, Bik peut surmonter l'effet protectrice contre l'apoptose de Bcl2b5 par rapport à Bakb5. Ce sont les premières évidences qu'une interaction binaire entre des membres de la famille Bcl2 au RE peut contrôler la mort cellulaire. La deuxième partie de cette étude était conçu pour déterminer le rôle de Bax/Bak et Bcl2 localisé uniquement au RE, dans la voie de signalisation initiée par p20Bap31. En utilisant des cellules provenant de l'épithélium rénale de souris nouveau-nés transformées par E1A/DNp53, soit de souche sauvage ou avec double knockout des gènes Bax et Bak, je démontre que l'expression ectopique de p20Bap31, mais non pas de Bik, peut initier une forme de mort cellulaire non-apoptotique indépendante de Bax/Bak qui ressemble la paraptose. Un résultat d'importance primordiale est qu'un délai de mort cellulaire attribué à Bcl2b5 s'avère dans l'absence de Bax et Bak, ce qui suggère un rôle pro-survie inattendu de Bcl2 au RE indépendant de Bax/Bak. Cette étude démontre une capacité de contrôler la mort cellulaire des membres de la famille Bcl2 par des interactions binaires au RE et une fonction inhibitrice de Bcl2 sur la mort cellulaire indépendante de Bax/Bak. De plus, une nouvelle voie de signalisation de mort cellulaire non-apoptotique initiée par p20Bap31 est identifiée.

Ero1α is a resident ER oxidase and is an important member of the oxidative protein folding machinery. It generates disulphide bonds de novo and donates them to protein disulphide isomerase (PDI), which in turn oxidises nascent substrate proteins within the ER. Ero1 activity must be tightly regulated for two key reasons: (i) it must maintain the balance of oxidised PDI to ensure oxidative protein folding can occur, but cannot be so active that the ER becomes hyperoxidising and dysfunctional, and (ii) Ero1 activity must be regulated to prevent the accumulation of hydrogen peroxide, a reactive oxygen species (ROS), within the ER. The regulation of Ero1α comes principally from 3 intramolecular disulphide bonds which are reduced by substrate upon activation, and re-oxidised upon inactivation by an unknown mechanism. Using an SDS-PAGE based assay we tested three hypotheses: that sulphenylation by Ero1α-produced hydrogen peroxide could induce re-oxidation; that an internal disulphide exchange mechanism could generate and distribute disulphide bonds within Ero1α; and that ER oxidoreductases could act to inactivate Ero1α. Having successfully expressed, purified and characterised a recombinant version of Ero1α, this was tested in a number of assays to address the above hypotheses. In vitro findings show that Ero1α is specifically and rapidly oxidised by ERp46 and PDI. Sulphenylation and internal disulphide exchange-mediated oxidation of Ero1α provided a comparatively slow and incomplete method of re-oxidation. In vivo results suggest that ERp46 and PDI may have implications in Ero1α activity regulation. Overexpression of several ER oxidoreductases had no effect on Ero1α re-oxidation after DTT challenge, whereas Ero1α oxidation was impaired slightly in PDI- ERp46 double knockdown cells. Depletion of PDI from cells results in the DTT-resistance of Ero1α, suggesting that Ero1α, PDI and glutathione are involved in an intricate mechanism of sensing and reacting to ER redox conditions. Two key ER oxidoreductases, PDI and ERp57, are oxidised in semi-permeabilised cells. Oxidation coincides with permeabilisation of the plasma membrane and the removal of cytosolic glutathione, directly implicating glutathione in the maintainence of the redox states of ER oxidoreductases. Oxidation during the permeabilisation of cells is an enzymatic process which is mediated in part by Ero1α. Semi-permeabilised cells harbour a more oxidising environment than do microsomes. This study contributes significantly to the research field by complementing several previously reported findings, as well as providing a novel investigation into the molecular regulation of Ero1α and its relationship with PDI and glutathione in the cell.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003.
Includes bibliographical references.
Membrane glycoproteins of the secretory pathway that cannot adopt their native conformation are targeted for dislocation from the endoplasmic reticulum (ER) membrane for subsequent degradation by the cytosolic proteasome. This thesis investigates factors influencing the catalyzed destruction of MHC class I molecules by the HCMV glycoproteins US2 and US 11 and the degradation of the model substrate TCRuc. The ER chaperone calnexin, implicated in glycoprotein folding, and the ER chaperones protein disulfide isomerase and Erolu, implicated in substrate disulfide bond formation, were examined for their roles in protein dislocation. By targeting these ER chaperones with siRNA constructs, the cellular levels of these ER chaperones were significantly reduced. Nevertheless, the rates of degradation of TCRct and the US2- and US 11-catalyzed destruction of MHC class I molecules were similar to wild-type cells. The Unfolded Protein Response (UPR) transcriptionally regulates ER chaperones and is essential in S. cerevisiae for efficient degradation of model ER substrates. In mammalian cells, neither the ATF6-dependent response nor the IREltc-XBP-1-dependent response of the UPR was found to be essential for efficient degradation of TCRc, US2-, or US 11-catalyzed destruction of MHC class I molecules. Interestingly, the ATF6 response, but not the IRElct-XBP-1 response, is essential for cellular viability. To better define the substrate requirements of US2 and US 11, the 30 residue cytoplasmic tail of MHC class I molecules was mutated. US2 can degrade MHC class I molecules with a cytoplasmic tail shortened to 10 residues or lengthened, by the fusion of GFP to the C-terminus, to several hundred residues.
(cont.) In contrast, US 11 only degrades MHC class I molecules possessing a cytoplasmic tail of 30 amino acids. These data support a model that US2 and US 11 act through distinct degradative mechanisms. To define modular functional domains of US2 and US 11, lumenal or cytoplasmic domains were reciprocally exchanged between the viral molecules and between each viral molecule and their degradation substrate, the HLA-A2 heavy chain. Most chimeric molecules were not targeted for degradation when expressed alone or with their complementary construct. However, the US 11 molecule in which the cytoplasmic tail was exchanged for that of HLA-A2 was rapidly dislocated from the ER to the cytosol. In addition, lumenal GFP possessing the transmembrane domain of US 11 and the cytoplasmic tail of HLA-A2 was rapidly dislocated from the ER to the cytosol. Mutation of the critical glutamine residue in the transmembrane domain of US 11 essential for destruction of MHC class I molecules resulted in a marked stabilization of the GFP construct.
by Patrick J. Stern.
Ph.D.