Cornelia Wilson

The neurotensin receptor-3 also known as sortilin is part of the new receptor family of vacuolar protein sorting 10 protein domain. Growing evidence show that the vacuolar protein sorting 10 protein domain family is implicated as a genetic risk factor for neurodegenerative diseases such as Alzheimer's disease, frontotemporal lobar degeneration, and Parkinson's disease, in addition to links associated with type 2 diabetes mellitus, lysosomal disorders, cardiovascular disease and atherosclerosis. In fact, sortilin expression is elevated in many human cell lines controlling the trafficking and release of neurotrophins. Hence, not surprisingly the imbalance of neurotrophin signaling is implicated in several human diseases. The fine regulation of the growth factor, brain derived nerve factor by sortilin mediates both neuronal and tumor cell survival, whereas in Alzheimer's disease sortilin mediated beta secretase-1 trafficking increases the cleavage of the beta-amyloid precur...

The transfer of exosomes containing both genetic and protein materials is necessary for the control of the cancer cell microenvironment to promote tumor angiogenesis. The nature and function of proteins found in the exosomal cargo, and the mechanism of their action in membrane transport and related signaling events are not clearly understood. In this study, we demonstrate, in human lung cancer A549 cells, that the exosome release mechanism is closely linked to the multifaceted receptor sortilin (also called neurotensin receptor 3). Sortilin is already known to be important for cancer cell function. Here, we report for the first time its role in the assembly of a tyrosine kinase complex and subsequent exosome release. This new complex (termed the TES complex) is found in exosomes and results in the linkage of the two tyrosine kinase receptors TrkB (also known as NTRK2) and EGFR with sortilin. Using in vitro models, we demonstrate that this sortilin-containing complex exhibits a contr...

ABSTRACT The endoplasmic reticulum (ER) is the site where most secretory proteins acquire their native conformation and gain access to the secretory pathway, and the cell surface. Proteins entering the secretory pathway are translocated across or inserted into the ER membrane either co-translationally or post-translationally through an aqueous pore in the ER membrane called the translocon (1). The emerging polypeptide chains may then interact with molecular chaperones to ensure their correct folding and assembly (2). Covalent modification of the polypeptide chain by the formation of native inter- and intrachain disulphide bonds stabilizes folded protein domains and cross-links subunits associated with oligomeric complexes. The ability of the ER-molecular chaperones and folding enzymes to recognize and bind to non-native substrates retains these proteins within the ER until they have reached their native state (3). The dissociation of the fully folded substrates from the ER chaperones facilitates the transport process, resulting in exit of the native protein from the ER. Therefore, the “quality-control” system of the ER allows export of only correctly folded and assembled proteins.

In eukaryotic cells, N-glycosylation is typically the most common protein modification that occurs in the endoplasmic reticulum (ER) lumen. N-glycosylation is facilitated by a large heterologous protein complex called the oligosaccharyltransferase (OST) that allows the attachment of a high mannose oligosaccharide from a dolichol pyrophosphate donor en bloc onto suitable asparagine residues of newly synthesized nascent chains during translocation into the ER lumen (1). While the complexity of the OST is highly conserved in eukaryotes, the role of its different subunits is poorly defined. We have investigated the function of three OST subunits, the ER translocon-associated component ribophorin I, and two isoforms of the presumptive catalytic subunit, STT3. We use a combination of siRNA-mediated knockdown of individual proteins combined with a semi-permeabilized mammalian cell system to provide a robust read out for OST subunit function during N-glycosylation of model substrates in vitro. This approach is equally applicable to the study of other cellular components.

ABSTRACT In eukaryotic cells, N-glycosylation is typically the most common protein modification that occurs in the ER lumen. N-glycosylation is facilitated by a large heterologous complex called the oligosaccharyltransferase (OST) which allows the attachment of a high mannose oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesised nascent chain during the translocation in the ER lumen. Defects in the process of N-glycosylation are often associated with a variety of diseases such as congenital disorders of glycosylation and Non-syndromic mental retardation. The focus of this work was based on our previous findings that some of the subunits of the OST may have alternative functions and play a role in the processing pathway of amyloid precursor protein (APP). We use a combination of methodologies developed during the project including RNA interference, Western blotting, and a fluorescent tripeptide assay to tackle these questions. Recent investigations reveal that depletion of some of the OST subunits, can affect APP processing leading to a reduction in both C-terminal fragments and full length N-glycosylated APP. The site of cleavage is of clinical relevance since Aβ peptides (Aβ 40-42) derived from γ-secretase cleavage of APP are major components of the amyloid plaques that are a characteristic brain lesion of individuals with Alzheimer's disease. Globally, depletion of some of the OST subunits has no effect upon N-glycosylation. The primary goal of this project is to establish if depleting these subunits would have any implication for Alzheimer's disease. We show for the first time that overexpression of some of the OST subunits causes an increase in the active γ-secretase complex particularly the N-terminal fragment of PS1 that is generated by endoproteolysis. The results from this study will enable us to understand better how the mammalian OST machinery attaches N-linked glycans to proteins and to understand the alternative roles of the OST subunits in Neurological diseases such as Alzheimer's disease and NSMR.

The endoplasmic reticulum (ER) is a major site of protein synthesis in eukaryotes. Newly synthesized proteins are monitored by a process of quality control, which removes misfolded or unassembled polypeptides from the ER for degradation by the proteasome. This requires the retrotranslocation of the misfolded proteins from the ER lumen into the cytosol via a pathway that, for some substrates, involves members of the recently discovered Derlin family. The Derlin-1 isoform is present as a dimer in the ER, and we now show that its dimerization is modulated by ER stress. Three distinct types of chemically-induced ER stress substantially reduce the levels of Derlin-1 dimer as assayed by both cross-linking and co-immunoprecipitation. The potential function of the different Derlin-1 populations with respect to ER quality control is investigated by analysing their capacity to associate with a misfolded membrane protein fragment. We show for the first time that Derlin-1 can associate with an aberrant membrane protein fragment in the absence of the viral component US11, and conclude that it is the monomeric form of Derlin-1 that interacts with this potential ER-associated degradation substrate. On the basis of these data we propose a model where the pool of active Derlin-1 in the ER membrane can be modulated in response to ER stress.

The expanding use of GSM devices has resulted in public concern. Chaperone-mediated autophagy (CMA) is a way for protein degradation in the lysosomes and increases under stress conditions as a cell defense response. α-synuclein, a CMA substrate, is a component of Parkinson disease. Since GSM might constitute a stress signal, we raised the possibility that GSM could alter the CMA process. Here, we analyzed the effects of chronic exposure to a low GSM-900MHz dose on apoptosis and CMA. Cultured cerebral cortical cells were sham-exposed or exposed to GSM-900MHz at specific absorption rate (SAR): 0.25W/kg for 24 h using a wire-patch cell. Apoptosis was analyzed by DAPI stain of the nuclei and western blot of cleaved caspase-3. The expression of proteins involved in CMA (HSC70, HSP40, HSP90 and LAMP-2A) and α-synuclein were analyzed by western blot. CMA was also quantified in situ by analyzing the cell localization of active lysosomes. 24 h exposure to GSM-900MHz resulted in ∼0.5°C temper...

Epidemiological data testifies the increasing incidence of Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Some associations were made between occidental lifestyle and development of these pathologies, moreover AD and T2DM are linked since each pathology is a causative risk factor for the other. Interestingly, autophagy, a catabolic pathway whose efficiency declines with age is importantly impaired in the affected tissues. Autophagy regulation is dependent of cell metabolic status and consequently on the 5'AMP-activated protein kinase (AMPK) and mammalian target of rapamycin signaling pathways. These pathways are altered with aging and molecular, pharmacological and physiological interventions increase lifespan in various organismal models and favours healthy aging diminishing the occurrence of age-related diseases such as diabetes, cancer, cardiovascular and neurodegenerative pathologies. Decreasing calorie intake has been known for a long time to have a beneficial effect on longevity and health. Some drug agonists of AMPK are known to mimic these effects such as metformin or resveratrol, a polyphenol extracted from plants and present in red wine, a component of the French paradox related diet. In this review, we present the epidemiological and pathogenesis links existing between AD and T2DM with an insight into the perturbations of the autophagic process highlighting the crucial role of the AMPK in development of age and metabolic related diseases. Hence, in a last part we will discuss the possible interventions susceptible to combat both T2DM and AD.