149 research outputs found
Luteinizing hormone receptor : Expression and post-translational regulation of the rat receptor and its ectodomain splice variant
No full textAbstractThe luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant.We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis.The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues.The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment.Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in Auditorium 5 of the Institute of Dentistry, on November 25th, 2005, at 12 noonAbstract
The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant.
We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis.
The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues.
The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment.
Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates
A novel fluorescent probe reveals starvation controls the commitment of amyloid precursor protein to the lysosome
No full textAlzheimer's disease is the most important cause of dementia but there is no therapy that has been demonstrated to stop or slow disease progression. Amyloid precursor protein (APP) is the source of amyloid-β (Aβ), which aggregates in Alzheimer's disease to form toxic oligomeric species. The endo-lysosomal system can clear APP and Aβ from the cell if these molecular species are trafficked through to the lysosome. Currently, there are no easy methods available for the analysis of lysosomal APP trafficking. We therefore generated a fusion protein (tandem-fluorescent, or tf-APP) that allows detection of changes in APP trafficking using accessible techniques such as flow cytometry. This permits rapid analysis or screening of genes and compounds that alter APP processing in the cell. Using our novel molecular probe, we determined that starvation induces trafficking of APP and APP-carboxy-terminal fragments (APP-CTFs) to the degradative endo-lysosomal network. In line with this finding, suppression of mTOR signalling using AZD8055 also strongly induced trafficking of APP to the endo-lysosomal system. Remarkably, activation of mTOR signalling via RHEB over-expression inhibited the starvation-induced autophagy but did not affect trafficking of tf-APP. These results show tf-APP can be used to determine how APP is trafficked through the lysosomal system of the cell. This molecular probe is therefore useful for determining the molecular mechanism behind the commitment of APP to the degradative pathway or for screening compounds that can induce this effect. This is important as clearance of APP and APP-CTF provides an important potential therapeutic strategy for Alzheimer's disease.Leanne K. Hein, Pirjo M. Apaja, Kathryn Hattersley, Randall H. Grose, Jianling Xie, Christopher G. Proud, Timothy J. Sargean
Degradation mechanism of a Golgi-retained distal renal tubular acidosis mutant of the kidney anion exchanger 1 in renal cells
No full textDistal renal tubular acidosis (dRTA) can be caused by mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1). Both recessive and dominant mutations result in mistrafficking of proteins, preventing them from reaching the basolateral membrane of renal epithelial cells, where their function is needed. In this study, we show that two dRTA mutants are prematurely degraded. Therefore, we investigated the degradation pathway of the kidney AE1 G701D mutant that is retained in the Golgi. Little is known about degradation of nonnative membrane proteins from the Golgi compartments in mammalian cells. We show that the kidney AE1 G701D mutant is polyubiquitylated and degraded by the lysosome and the proteosome. This mutant reaches the plasma membrane, where it is endocytosed and degraded by the lysosome via a mechanism dependent on the peripheral quality control machinery. Furthermore, we show that the function of the mutant is rescued at the cell surface upon inhibition of the lysosome and incubation with a chemical chaperone. We conclude that modulating the peripheral quality control machinery may provide a novel therapeutic option for treatment of patients with dRTA due to a Golgi-retained mutant.Carmen Y. Chu, Jennifer King, Mattia Berrini, Alina C. Rumley, Pirjo M. Apaja, Gergely L. Lukacs, R. Todd Alexander and Emmanuelle Corda
Regulation of mTOR Signalling and Protein Synthesis via Parkinson's Disease Risk Factors UCHL1 and LRRK2
No full textParkinson's disease (PD) is an age-related, neurodegenerative disorder characterised by the loss of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). Most often, the formation of Lewy Bodies (LBs) occurs, LBs being -synuclein-rich protein aggerates, located intracellularly in surviving DA neurons. The loss of DA neurons of the SNpc results in debilitating motor symptoms for PD patients that, with the progression of the disease, have a significant effect on daily activities from eating to walking. To date, the causal, molecular mechanism that results in the loss of DA neurons of the SNpc in PD patients is poorly defined. However, mutations in as many as 18 genes have been associated with the onset of PD; termed the PARK genes, they indicate that specific cellular pathways associated with mitochondrial clearance (mitophagy) and maintenance, and potentially protein synthesis, are involved in the pathophysiology of PD. Two of the PARK genes are ubiquitin C-terminal hydrolase L1 (UCHL1) and leucine-rich repeat kinase 2 (LRRK2) both of which have interesting links to signalling through the mechanistic target of rapamycin (mTOR) a protein kinase. Cellular growth, autophagy, including mitophagy, and protein synthesis are all potently regulated by mTOR, suggesting UCHL1 and LRRK2 may act through mTOR to contribute to the development of PD. The regulation of mTOR signalling by UCHL1 has so far only been investigated in the context of cancer, with no data on effects of the PD-associated mutant, UCHL1I93M. Hence, this thesis, in part, aimed to understand whether and how UCHL1I93M interacts with the mTOR pathway, with the aim of further understanding the cellular mechanisms perturbed in PD. Unfortunately, it was not possible to reproduce previously published effects and it is apparent that UCHL1 does not regulate mTOR signalling. One well-studied substrate for mTOR complex 1 (mTORC1) is eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), a negative regulator of protein synthesis. Curiously, LRRK2 has been reported to phosphorylate 4E-BP1, as well as various ribosomal proteins. Functional inhibition of 4E-BP1 by mTORC1-mediated phosphorylation promotes protein synthesis. For this reason, this thesis also aimed to investigate the effects of LRRK2 on these mTORC1 targets. In this study, it was found that LRRK2 does appear to regulate mTORC1 signalling, although the mechanism by which LRRK2 has this effect remained elusive. Nonetheless, in HEK293 cells, overexpression of LRRK2WT or LRRK2G2019S, the PDassociated mutant, enhanced the serum-induced phosphorylation of 4E-BP1 and, in MEFs, LRRK2G2019S knock-in induced a similar effect but on a different mTOR target, RPS6. Ultimately, it was concluded that UCHL1 does not act through mTORC1 signalling to induce PD. However, LRRK2 was found to affect the phosphorylation of 4E-BP1 and RPS6, but the mechanism by which LRRK2 regulates these mTOR targets and the consequences for protein synthesis and the PD phenotype remain subjects for future work.Thesis (MPhil) -- University of Adelaide, School of Biological Sciences, 202
Luteinizing hormone receptor:expression and post-translational regulation of the rat receptor and its ectodomain splice variant
No full textAbstract
The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant.
We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis.
The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues.
The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment.
Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates
Alpha-B-Crystallin overexpression is sufficient to promote tumorigenesis and metastasis in mice
No full textData source: Supplementary Information, https://doi.org/10.1186/s40164-022-00365-zBackground: αB-Crystallin is a heat shock chaperone protein which binds to misfolded proteins to prevent their aggregation. It is overexpressed in a wide-variety of cancers. Previous studies using human cancer cell lines and human xenograft models have suggested potential tumor promoter (oncogene) roles for αB-Crystallin in a widespectrum of cancers. Methods: To determine the causal relationship between CRYAB overexpression and cancer, we generated a Cryab overexpression knock-in mouse model and monitor them for development of spontaneous and carcinogen (DMBA)- induced tumorigenesis. In order to investigate the mechanism of malignancies observed in this model multiple techniques were used such as immunohistochemical characterizations of tumors, bioinformatics analysis of publically available human tumor datasets, and generation of mouse embryonic fbroblasts (MEFs) for in vitro assays (clonogenic survival and migration assays and proteome analysis by mass-spectrometry). Results: This model revealed that constitutive overexpression of Cryab results in the formation of a variety of lethal spontaneous primary and metastatic tumors in mice. In vivo, the overexpression of Cryab correlated with the upregulation of epithelial-to-mesenchymal (EMT) markers, angiogenesis and some oncogenic proteins including Basigin. In vitro, using E1A/Ras transformed MEFs, we observed that the overexpression of Cryab led to the promotion of cell survival via upregulation of Akt signaling and downregulation of pro-apoptotic pathway mediator JNK, with subsequent attenuation of apoptosis as assessed by cleaved caspase-3 and Annexin V staining. Conclusions: Overall, through the generation and characterization of Cryab overexpression model, we provide evidence supporting the role of αB-Crystallin as an oncogene, where its upregulation is suffcient to induce tumors, promote cell survival and inhibit apoptosis.Behnam Rashidieh, Amanda Louise Bain, Simon Manuel Tria, Sowmya Sharma, Cameron Allan Stewart, Jacinta Ley Simmons, Pirjo M. Apaja, Pascal H. G. Duijf, John Finnie, and Kum Kum Khann
Reprogramming endo-lysosomal proteostasis disease stress by UBR1- and arginylation-driven endophagy and autophagy protein quality control
No full textProtein quality control (PQC) is a conformational surveillance system critical to maintaining native protein composition in the cell. However, PQC mechanisms at the endo-lysosomal pathway especially toward membrane proteins and during cumulative endo-lysosomal stress are incompletely understood. We recently identified the ubiquitin ligase UBR1 as a PQC E3 ubiquitin-ligase for endosomal and/or cytosolic Ca2+-increase mediated proteostasis disease stress. As a consequence of the endosomal stress and/or cytosolic Ca2+-increase, the QC pathway using selective endosomal autophagy (endophagy) and autophagy was activated for ubiquitinated and arginylated UBR1-SQSTM1/p62 cargoes. In turn, the loss of UBR1, arginylation or both evoke endo-lysosomal pathway stress. Our data suggest that UBR1 with arginylation-dependent endophagy and autophagy is required during proteostasis perturbations and highlight the importance of UBR1 in stress-induced autophagy QC with implications for various human diseases
Chaperones rescue the energetic landscape of mutant CFTR at single molecule and in cell
No full textMolecular chaperones are pivotal in folding and degradation of the
cellular proteome but their impact on the conformational dynamics of
near-native membrane proteins with disease relevance remains unknown.
Here we report the effect of chaperone activity on the functional
conformation of the temperature-sensitive mutant cystic fibrosis channel
(Delta F508-CFTR) at the plasma membrane and after reconstitution into
phospholipid bilayer. Thermally induced unfolding at 37 degrees C and
concomitant functional inactivation of Delta F508-CFTR are partially
suppressed by constitutive activity of Hsc70 and Hsp90
chaperone/co-chaperone at the plasma membrane and post-endoplasmic
reticulum compartments in vivo, and at singlemolecule level in vitro,
indicated by kinetic and thermodynamic remodeling of the mutant gating
energetics toward its wild-type counterpart. Thus, molecular chaperones
can contribute to functional maintenance of Delta F508-CFTR by reshaping
the conformational energetics of its final fold, a mechanism with
implication in the regulation of metastable ABC transporters and other
plasma membrane proteins activity in health and diseases
Applying DATEMATS Methods and Tools to Experimental Wood-Based Materials: Materiality in an Ideation Process
No full textPublisher Copyright: © 2023, The Author(s).This chapter provides an overview of the DATEMATS Experimental wood-based materials workshop and student projects, and a more detailed description of one team's ideation process. The workshop was held at the Chemarts facilities of Aalto University, Finland, in January 2022. A total of 19 students from four different universities and from the fields of design and engineering participated in a five-day workshop creating innovative applications for interior panels made of cellulose waste. The company challenge was given by Honext from Spain.Peer reviewe
Le mage comme métaphore de l'écrivain: le cas de Joséphin Péladan
No full textJoséphin Péladan, author of Décadence Latine and also founder of the Rose+Croix du Temple et du Graal Confraternity, represents from various angles the symbol of that cultural atmosphere which characterizes the end of the nineteenth century. As he stigmatizes the moral and cultural decline of his contemporaries, Péladan creates the character of Mérodack the Magus, described as a supreme being that arises above the bourgeois mediocrity and who, thanks to a self-sublimation process based on will-power, according to the outlook already traced by Eliphas Lévi, not only can redeem himself, but also can show the direction for a shared redemption. However, as a matter of fact, the solipsistic and narcissistic attitude of the Magus ends by overwhelming any social temptation. In this path which shows preferential connections between literature and occultism, the Magus, former other self of the author, his double in the mirror, as their physical resemblance and the interchange of names prove, rises to the metaphor of the writer: both committed to a self-referring dimension, they transgress the bourgeois rules of productivity by changing magic and writing into an ascesis, a self-commemorative operation only consecrated to the ego. This idea of magic, conceived as a metaphor of writing as well, as a hermetic and mystic operation appears, in conclusion as the utmost accomplishment, daintily decadent, of Mallarmé's poetry conception, just a supreme act, which is to find the reason of his own existence solely in itself
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