THE PROTEOSTASIS OF THE ENDOPLASMIC RETICULUM AND THE ACTIVATION OF THE UNFOLDED PROTEIN RESPONSE PATHWAY IN VIVO

ABSTRACT Background. The protein components of eukaryotic cells face acute and chronic challenges to normal folding, refolding and function owing to a constant barrage of physical, metabolic and environmental stresses. Eukaryotic protein homeostasis, or proteostasis, enables healthy cell adaptation during development and protects against aging and diseases. Proteostasis refers to controlling the concentration, conformation, binding interaction and location of individual proteins making up the proteome by readapting the innate biology of the cell, often through transcriptional and translational changes. The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways, cumulatively called the unfolded protein response (UPR). The UPR activation triggers an extensive transcriptional and translational response, which adjusts the ER protein folding capacity according to needs. As such, the UPR constitutes one of the signaling pathways that regulates the capacity and composition of the proteostasis network according to the changing of the ER folding capacity. Previous work. My initial work, described in chapter 1.2.1 shows that the level of cellular energy is important for protein folding and disaggregation and thus can affect folding and repair processes. The proteostasis network is not only highly adaptable, enabled by the influence of multiple cell stress signaling pathways, but also can be quite distinct in each cell type. In chapter 1.2.2, by using as a cellular model the plasma cells differentiation, I could underline the role of the UPR signaling and proteasomal degradation in orchestrating the architectural and functional changes of the cells and balancing the proteostasis capacity. Recent studies suggest that lack/decrease of O2 perturbs the ER homeostasis. Indeed the ER has emerged as a cellular compartment that depends on O2 for oxidative folding of secretory and transmembrane proteins and that mediates the O2 signaling that is important for the survival and function of hypoxic cells. In vitro studies have shown that hypoxia triggers the UPR. However, in vivo and in vitro situations are indeed likely to diverge substantially with respect to parameters such as metabolic activity, O2 utilization and cell division rates, features that are predicted to vary the cell sensitivity to ER stress. Thus, despite a recognized role for hypoxia on UPR, few data exist on the effects of hypoxia in various organs in vivo. Aims. My study includes three aims: First, we will test if the hypoxic stress in vivo acts as a modifier that affects the activation of specific branches of the UPR in different tissues. Second, to get a better insight into the role of the UPR during low oxygen availability in tissues, we will test whether the UPR activation depends from the severity of the hypoxic stress. Third, we aim at delineating signaling circuits that control the capacity and composition of the proteostasis network through transcriptional and post transcriptional mechanisms to balance the ER homeostasis Results. I analyzed the effect of the hypoxic stress on the proteostasis network. Changes in O2 levels alter the ability of the cells to handle the proteostasis load with some differences between the cells type studied. Hepatocytes and myocytes respond to hypoxia by increasing their degradation activity as to increase the proteostasis capacity. While the hepatocytes activate an UPR-dependent apoptosis and are able to balance between apoptotic death and protein synthesis, in the myocytes the protein synthesis remains sustained under low oxygen availability while the UPR \u2013dependent apoptosis could not be detected. Conclusion. This studies underlined several features of the ER- proteostasis. First, the proteostasis network is adaptable and able to fine tune the UPR signaling pathway in response to stress. Second, different cells have varying proteostasis capacities reflected in the composition and concentrations of their proteostasis components. Third, within a given cell type, the proteostasis does not possess significant excess capability, rater it is finely tuned and offers just enough facility for the protein folding load. Therefore, by setting the proteostasis boundary as a threshold for generating folded and functional proteins, the proteostasis network can create and maintain functional proteins in response to the local environment

Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B.

Background & aims In chronic HBV infection, mitochondrial functions and proteostasis are dysregulated in exhausted HBV-specific CD8 T cells. To better characterise the potential involvement of deregulated protein degradation mechanisms in T cell exhaustion, we analysed lysosome-mediated autophagy in HBV-specific CD8 T cells. Bioactive compounds able to simultaneously target both mitochondrial functions and proteostasis were tested to identify optimal combination strategies to reconstitute efficient antiviral CD8 T cell responses in patients with chronic HBV infection. Methods Lysosome-mediated degradation pathways were analysed by flow cytometry in virus-specific CD8 T cells from patients with chronic HBV infection. Mitochondrial function, intracellular proteostasis, and cytokine production were evaluated in HBV-peptide-stimulated T cell cultures, in the presence or absence of the polyphenols resveratrol (RSV) and oleuropein (OLE) and their metabolites, either alone or in combination with other bioactive compounds. Results HBV-specific CD8 T cells from patients with CHB showed impaired autophagic flux. RSV and OLE elicited a significant improvement in mitochondrial, proteostasis and antiviral functions in CD8 T cells. Cytokine production was also enhanced by synthetic metabolites, which correspond to those generated by RSV and OLE metabolism in vivo, suggesting that these polyphenols may also display an effect after transformation in vivo. Moreover, polyphenolic compounds improved the T cell revitalising effect of mitochondria-targeted antioxidants and of programmed cell death protein 1/programmed cell death ligand 1 blockade. Conclusions Simultaneously targeting multiple altered intracellular pathways with the combination of mitochondria-targeted antioxidants and natural polyphenols may represent a promising immune reconstitution strategy for the treatment of chronic HBV infection. Lay summary In chronic hepatitis B, antiviral T lymphocytes are deeply impaired, with many altered intracellular functions. In vitro exposure to polyphenols, such as resveratrol and oleuropein, can correct some of the deregulated intracellular pathways and improve antiviral T cell function. This effect can be further strengthened by the association of polyphenols with antioxidant compounds in a significant proportion of patients. Thus, the combination of antioxidants and natural polyphenols represents a promising strategy for chronic hepatitis B therapy

Effetti dell’Urolitina A sulla funzionalità muscolare

L’Urolitina, uno dei metaboliti derivati dal metabolismo degli ellagitannini, é stata studiata in vitro e in vivo per le sue funzioni a livello del muscolo scheletrico correlate ad un miglioramento della funzione mitocondriale. Il metabolismo mitocondriale é un’importante fonte energetica per il muscolo poiché la sua funzione principale é quella di generare ATP. Studi in modelli animali hanno evidenziato che la somministrazione di questo composto migliora la funzione mitocondriale e la catena respiratoria promuovendo l’induzione della biogenesi e della mitofagia impedendo così l’accumulo di mitocondri disfunzionali. Studi in umano hanno evidenziato come l’Urolitina A, riducendo il declino correlato all’età, possa essere un’ottima modalità per accompagnare l‘invecchiamento e promuovere una buona funzionalità muscolare. L’integrazione a lungo termine ha avuto un’azione positiva sulla resistenza muscolare. L’Urolitina A, agendo sull'attività mitocondriale, risulta pertanto un approccio promettente per contrastare il declino muscolare correlato all'età

Thioredoxin Downregulation Enhances Sorafenib Effects in Hepatocarcinoma Cells

Sorafenib is the first-line recommended therapy for patients with advanced hepatocarcinoma (HCC) in de-differentiation stage (presenting epithelial–mesenchymal transition, EMT). We studied the role of the thioredoxin system (Trx1/TrxR1) in the sensitivity or resistance of HCC cells to the treatment with Sorafenib. As a model, we used a set of three established HCC cell lines with different degrees of de-differentiation as occurs in metastasis. By quantitative proteomics, we found that the expression levels of Trx1 and TrxR1 followed the same trend as canonical EMT markers in these cell lines. Treatment with Sorafenib induced thiol redox reductive changes in critical elements of oncogenic pathways in all three cell lines but induced drastic proteome reprograming only in HCC cell lines of intermediate stage. Trx1 downregulation counteracted the thiol reductive effect of Sorafenib on Signal Transducer and Activator of Transcription 3 (STAT3) but not on Mitogen-Activated Protein Kinase (MAPK) or Protein Kinase B (Akt) and transformed advanced HCC cells into Sorafenib-sensitive cells. Ten targets of the combined Sorafenib–siRNATrx1 treatment were identified that showed a gradually changing expression trend in parallel to changes in the expression of canonical EMT markers, likely as a result of the activation of Hippo signaling. These findings support the idea that a combination of Sorafenib with thioredoxin inhibitors should be taken into account in the design of therapies against advanced HCC.Ministerio de Economía y Competitividad BFU2016-80006-PInstituto de Salud Carlos III (ISCIII) PI13/00021 y PI16/00090Junta de Andalucía Consejería de Economía, Innovación, Ciencia y Empleo BIO-0216 y CTS-6264Junta de Andalucía Consejería de Igualdad, Salud y Políticas Sociales PI-00025-2013 y PI-0198-201

Espressione e localizzazione del DNAJC13, una proteina coinvolta nella malattia di Parkinson

La malattia di Parkinson (MP) è un disturbo neurodegenerativo contraddistinto dalla perdita di neuroni nella substantia nigra pars compacta (SNpc) che causa carenza di dopamina striatale. Le cause e i meccanismi alla base di questa malattia non sono ben noti ma si è visto che mutazioni a carico della proteina DNAJC13 comportano un alterato traffico vescicolare contribuendo alla manifestazione della MP. Lo scopo di questo studio è stato caratterizzare l’espressione di DNAJC13 tramite tecniche di western blot e di immunocitochimica. Nello specifico è stato utilizzato un plasmide condiviso dal ricercatore giapponese Kiyotoshi Sekiguchi per esprimere la proteina DNAJC13 legata ad un tag GFP. Dopo averlo amplificato con il protocollo MIDI, abbiamo controllato la sua integrità confrontando i risultati della digestione con HindIII in silico con quelli sperimentali; è stata poi osservata la sua espressione nelle cellule HEK293T. Successivamente le cellule sono state lisate e il DNAJC13-GFP è stato purificato per verificare il peso molecolare della proteina attraverso un western blot. Infine, è stato eseguito un trattamento con il fattore di crescita dell’epidermide (EGF) perché da letteratura sembra che il DNAJC13 sia coinvolto nella sua endocitosi. Le cellule sono state sottoposte al protocollo di immunocitochimica e poi analizzate al microscopio a fluorescenza per risalire ad eventuali differenze di localizzazione della proteina in seguito al trattamento con l’EGF.Parkinson's disease (PD) is a neurodegenerative disorder characterised by by the loss of neurons in the substantia nigra pars compacta (SNpc) causing striatal dopamine deficiency. The causes and mechanisms underlying this disease are not well known but it has been shown that mutations in the DNAJC13 protein lead to altered vesicular trafficking, contributing to the manifestation of MP. The aim of this study was to characterise the expression of DNAJC13 by means of western blot and immunocytochemistry techniques. Specifically, the following was used a plasmid shared by Japanese researcher Kiyotoshi Sekiguchi to express the DNAJC13 protein bound to a GFP tag. After amplifying it using the MIDI protocol, we checked its integrity by comparing the results of digestion with HindIII in silico with those experimental ones; its expression was then observed in HEK293T cells. Subsequently, the cells were lysed and the DNAJC13-GFP was purified to verify the molecular weight of the protein by western blot. Finally, treatment was performed with epidermal growth factor (EGF) because it appears from the literature that DNAJC13 is involved in its endocytosis. The cells were subjected to the immunocytochemistry protocol and then analysed under a fluorescence microscope to trace any differences in localisation of the protein following treatment with EGF

Characterization of patients with mitochondrial disease: assessment of the pathological phenotype associated with genes involved in mitochondrial quality control and dynamics

Summary: The maintenance of healthy and functional mitochondria is essential for cellular homeostasis; a first check point is provided by the organelle itself through the mitochondrial quality control and through the mitochondrial dynamics. Mitochondrial dynamics involve the Drp1 protein, a large dynamin-like GTPases encoded by DNM1L gene, that is responsible for fission of mitochondria. Mutations in DNM1L gene have been associated with several neurological disorders (Schmid et al. 2019). Furthermore, one of the main players of the mitochondrial quality control is the Lon protease encoded by LONP1 gene, involved in mitochondrial proteostasis and in the maintenance of mitochondrial DNA. Mutations in LONP1 were associated with a multisystem disorder called CODAS (Cerebral, Ocular, Dental, Auricular, Skeletal) syndrome (Strauss et al. 2015) and, more recently, with a classical mitochondrial disease phenotype (Peter et al. 2018; Nimmo et al. 2019). Through the use of Next Generation Sequencing (NGS) technology, we identified new mutations in genes involved in mitochondrial quality control and dynamics. In five patients, we identified de novo dominant DNM1L variants, two of which have been never reported. Patients’ fibroblast displayed defects in mitochondrial morphology; interestingly, we observed, in muscle biopsies, changes in mitochondrial distribution. To date, no peculiar histochemical alterations have been reported in DNM1L-mutated patients and this can represent a diagnostic tool. In one patient, we found three different mutations in LONP1, never described before. These variants cause both energy defects and alterations in mitochondrial network too

Validazione dell'interazione tra le proteine LRRK2 e TRIM32 e implicazione nella malattia di Parkinson

La Malattia di Parkinson (MP) è la seconda malattia neurodegenerativa più comune. Nelle forme familiari sono state caratterizzate varianti del gene LRRK2. Nonostante i meccanismi alla base della malattia non siano ancora chiari, la patogenicità di tali varianti gain of function è ben nota. È necessario, perciò, chiarire il ruolo fisiologico della proteina LRRK2 e capire come questa sia coinvolta nella patogenesi della malattia. Fondamentale per caratterizzare i pathways che la riguardano è studiare le proteine con cui interagisce. Analisi preliminari di proteomica effettuate nel laboratorio dove ho svolto il tirocinio hanno evidenziato TRIM32, una E3-ubiquitin ligasi, come possibile interattore di LRRK2; quindi, la sua attività potrebbe contribuire alla patogenesi nella MP. L’obiettivo di questo studio è quello di validare l’interazione tra LRRK2 e TRIM32. Per fare ciò, sono state ottenute linee cellulari esprimenti LRRK2, con tag Flag, wild-type e mutata. Attraverso l’immunoprecipitazione con resina anti-Flag e l’analisi di Western Blot è stata validata l’interazione tra le due proteine. Inoltre, in seguito alla quantificazione dei risultati ottenuti, l’entità dell’interazione tra TRIM32 e LRRK2 mutata si è rivelata differente rispetto alla condizione fisiologica

Il ruolo di USP14 nella regolazione della traslocazione di TFEB al nucleo

L’autofagia e il sistema ubiquitina-proteasoma (UPS) sono i principali meccanismi responsabili dell’omeostasi cellulare negli eucarioti. I due processi utilizzano l’ubiquitinazione come segnale di degradazione per controllare la qualità intracellulare. L’UPS si occupa della degradazione delle proteine di breve durata danneggiate o mal ripiegate, mentre l’autofagia elimina le proteine di lunga durata, organelli disfunzionali e parassiti intracellulari, per esempio i batteri

Mass spectrometry-based proteomics strategies to define protein NEDDyltion

124 p.Las modificaciones post-traduccionales reguladas por la ubiquitina y la familia de la ubiquitina estánimplicadas en diversas funciones vitales en las células eucariotas. Entre todas las proteínas de la familiade la ubiquitina, NEDD8 es la que comparte mayor similitud con la ubiquitina tanto en su secuenciacomo en su estructura. A pesar de esta similitud, NEDD8 tiene su propio conjunto de enzimas que resultaen una distinta cascada de conjugación. Las culinas, forman parte de la familia de ligasas de ubiquitinaCRLs (Culina-RING) y representan los sustratos mejor caracterizadas de NEDD8, por ello, la mayoría delos estudios sobre NEDDilación se han enfocado principalmente en la regulación y la degradación deproteínas ubiquitiladas. Recientemente, otros sustratos relevantes de NEDD8 han sido identificados locual sugiere que la NEDDilación está vinculada en muchos más procesos biológicos esenciales para lacélula. De hecho, NEDD8 responde a situaciones de estrés celular en comunicación cruzada con laubiquitina, sin embargo, todavía se desconoce el papel de la NEDDilación en respuesta al estrés. Laimportancia de la NEDDilación se ha sustentado gracias su inhibidor, MLN4924, usado exitosamente enel tratamiento de ciertos tipos de cáncer. En consecuencia, la identificación de nuevos sustratos deNEDD8 potencialmente ayudará a ampliar nuestra comprensión sobre los mecanismos por el cual suinhibición induce efectos anti-tumorales. No obstante, el conocimiento de las proteínas NEDDiladas estámuy limitado debido, en gran parte, a que las recientes técnicas en espectrometría de masa no permitendistinguir entre la NEDDylación y la ubiquitilación ya que ambas proteínas liberan la misma señal dobleglicina (GG) después de la digestión con tripsina del péptido modificado. En este trabajo nos enfocamosen el desarrollo de nuevas estrategias aplicadas en proteómica para resolver el problema que supone laidentificación a gran escala de las proteínas NEDDiladas. Con nuestros avances hemos logrado no solodistinguir sustratos específicos de NEDD8 sino también los sitios de modificación. Además, por mediode métodos de cuantificación en combinación con espectrometría de masas, hemos podido caracterizar elpapel de la NEDDilación en situaciones de conjugación canónica y atípica explorando así más allá de supapel homeostático como regulador de las CRLs.CRBM :Centre de Recherche en Biologie Celulaire de Montpellier. InbioMed : Investigación en medicina regenerativa ITAV : Centre Pierre Potier CONACYT :Consejo Nacional de Ciencia y Tecnología COST European Cooperation in Science and Technology Proteostasi