O proteassoma 26S é uma estrutura em forma de barril com um núcleo catalítico 20S que é flanqueado por tampas 19S em ambos os lados. Nosso grupo tem demonstrado que o proteassoma 26S é crítico para o desenvolvimento e sobrevivência do Schistosoma mansoni, sendo que mais de 95% dos casais de vermes tratados com o inibidor de proteassoma MG132 apresentaram alteração na postura dos ovos e viabilidade. O tampão 19S é o complexo regulador e funciona no desdobramento e na desubiquitinação das proteínas, antes de sua entrada no complexo 20S, usando enzimas desubiquitinadoras (DUBS). Recentemente, foi demonstrado que a inibição das DUBS, UCHL5 e USP14, que estão reversivelmente ligadas à partícula 19S do complexo 26S, chamada b-AP15, resulta em autofagia, seguida de morte celular, pois leva uma variedade de células de mamíferos ao estresse oxidativo. O objetivo geral deste trabalho foi avaliar o efeito da droga sobre o desenvolvimento reprodutivo de S. mansoni e como possível alvo terapêutico, tendo como objetivos específicos determinar os seus efeitos: na inibição da oviposição in vitro de casais de parasitas Schistosoma mansoni através da contagem dos ovos e expressão do gene p14; avaliar se os órgãos reprodutores dos parasitas sofreram alguma modificação estrutural; se a substância utilizada leva a autofagia; se o tratamento dos casais de parasita com a droga leva a apoptose, ocasionada pela ativação de caspase-3. Este estudo é o primeiro a documentar o papel da droga b-AP15 como um agente esquistossomicida, pois desencadeia alterações ultraestruturais em casais de vermes de S. mansoni. Os seguintes métodos foram utilizados para analisar as alterações: Microscopia Eletrônica de Transmissão (MET), Varredura (MEV) e Confocal; ensaio quantitativo colorimétrico baseado no brometo de 3- (4,5-dimetiltiazol-2-il) -2,5-difenil tetrazólio (MTT), atividade do proteassoma através do substrato Suc-Leu-Le-Al-Tyr-AMC específico para atividade de quimotripsina-like; western blotting; Reação de Polimerase em Cadeia em tempo real e TUNEL. Foram testadas várias concentrações do fármaco (0,2; 0,4; 0,8; 1,6; 3,2 até 50 µM), sendo que a partir de 1,6 µM de b-AP15 ocorreu a inibição da produção de ovos dos casais de parasitas tratados in vitro por 24 h, não havendo alteração da viabilidade, mas mostrou alterações a partir da dosagem de 3,2 µM. A partir de 1,6 µM ocorreram alterações celulares e tegumentares e vermes adultos tratados com 50 µM estavam mortos. O Western blotting mostrou acúmulo de proteínas poliubiquitinadas de alto peso molecular na presença de 1,6µM, havendo mudanças na atividade quimotripsina-like do proteassoma 20S. Os resultados mostraram que o b-AP15 altera a oviposição, a viabilidade e leva à morte de casais de parasitas, reforçando a hipótese de que o sistema ubiquitina/ proteassoma e que as enzimas deubiquitinadoras ligadas a partícula 19S são essenciais para a biologia de S. mansoni. / The 26S proteasome is a barrel structure with a catalytic core 20S that is flanked by 19S caps on both sides. Our group has been showed that 26S proteasomes are critical for Schistosoma mansoni development and survival, being that more than 95% of worms pairs treated with the proteasome inhibitor MG132 showed alteration on egg laying and viability. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14, which are reversibly bound to the 19S particle of the 26S complex, called b-AP15 , results in cell death because it leads to autophagy, followed by cell death in a variety of mammalian cells. The general objective of this work was to evaluate the effect of the drug on the reproductive development of S. mansoni and as a possible therapeutic target. The specific objectives to determine its effects: in inhibiting the in vitro oviposition of couples of Schistosoma mansoni parasites by counting eggs and p14 gene expression; evaluate whether the reproductive organs of the parasites have undergone some structural modification; if the substance used leads to autophagy; if the treatment of the parasite couples with the drug leads to apoptosis, caused by the activation of caspase-3. This study is the first to document the role of the b-AP15 drug as a schistosomicidal agent, as it triggers ultrastructural changes in couples of S. mansoni worms. The following methods were used to analyze the changes: Transmission Electron Microscopy (MET), Scanning (SEM) and Confocal; Colorimetric assay based on 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), proteasome activity through Suc-Leu-Le-Al-Tyr-AMC substrate specific for chymotrypsin-like activity; western blotting; Polymerase chain reaction in real time and TUNEL. Various concentrations of the drug (0.2, 0.4, 0.8, 1.6, 3.2 to 50 ?M) were tested. 1.6 ?M b-AP15 occurred the inhibition of egg production of couples of parasites treated in vitro for 24 h, with no change in viability, but showed changes from the dosage of 3.2 ?M. Cell and tegumentary changes occurred in 1.6 ?M and adult worms treated with 50 ?M were dead. Western blotting showed accumulation of high molecular weight polyubiquitinated proteins in the presence of 1.6?M, with changes in the chymotrypsin-like activity of the 20S proteasome. The results showed that b-AP15 alters the oviposition, viability and leads to the death of couples of parasites, reinforcing the hypothesis that the ubiquitin / proteasome system and the deubiquitinating enzymes bound to the 19S particle are essential for the biology of S. mansoni.

DNA is constantly subjected to chemical and mechanical damage. The ability to repair the lesions sustained is essential for all life. Double stranded DNA (dsDNA) breaks are especially toxic as both antiparallel strands of DNA are severed. The most high fidelity mechanism available to repair this damage is homologous recombination, a mechanism that uses homology from the sister chromatid to replace any lost information. Key proteins involved in maintaining genomic stability this way are conserved in all domains of life. One such component is the Mre11/Rad50 complex that is involved in the initial recognition of damage and recruitment of subsequent repair factors. Understanding the function of this DNA repair complex and any associated proteins has implications for human cancers and aging. The proteins of thermophilic archaea present an excellent opportunity to study these systems in a robust, tractable and eukaryote-like system. Archaea are in many ways biochemically unique, for example they are the only domain capable of methanogenesis. However archaea share a high level of homology with eukaryotes in many essential cellular processes such as DNA replication, homologous recombination and protein degradation. In thermophilic archaea the mre11/rad50 genes are clustered in an operon with the herA/nurA genes that form a helicase/nuclease complex. This has lead to speculation that the four proteins work together during homologous recombination to produce the 3' overhangs required by RadA to identify homology. As part of this investigation I have performed extensive bioinformatic searches of a variety of archaeal/bacterial systems. These analyses have revealed operonic linkages to other known recombinational helicase/nucleases, such as AddAB and RecBCD. These genomic linkages are especially prevalent in thermophilic organisms suggesting their functional relevance is particularly acute in organisms exposed to a high amount of genomic stress. Comparison of the evolutionary trees, constructed for each protein, makes a single genomic linkage event the most likely scenario, but cannot definitively exclude other possibilities. Exhaustive attempts were made to demonstrate an interaction between Mre11/Rad50 and HerA/NurA. Despite analysis by nickel/cobalt pulldown, immunoprecipitation, analytical gel filtration, ITC and OCTET an interaction could not be confirmed or definitively dismissed. However in the process an interesting Rad50 tetrameric assembly was identified and attempts were made to crystalize it. Hexameric helicases and translocases are key to the replication and DNA packaging of all cellular life and multiple viruses. The hexameric translocase HerA is a robust model for investigating the common features of multimeric ATPases as it is extremely stable and experimentally tractable. Here it is revealed that HerA exists in a dynamic equilibrium fluctuating between hexameric and heptameric forms with rapidly interchanging subunits. This equilibrium can be shifted to heptamer by buffering conditions or towards the hexamer by the physical interaction with the partnering nuclease NurA, raising the possibility that these alternate states may play a role in translocase assembly or function. A novel C-terminal brace, (revealed by a collaborative crystallographic structure) is investigated; as well as stabilizing the assembly, this brace reaches over the ATPase active site of its neighbouring subunit. It is seemingly involved in the conversion of energy generated by ATP hydrolysis into physical movement in the central channel of the hexamer. The regulation of homologous recombination is extremely important to prevent aberrant activity, resulting in mutations and genome reorganization. In eukaryotic organisms, it is well established that post-translational modifications and protein turnover at the proteosome play important roles in this control. In particular, there is significant interest currently in the ubiquination-proteasome destruction pathway as a mechanism for extracting DNA repair components from chromatin at the termination of the DNA repair process. To date no Ubiquitin proteins have been identified in the Archaea, however related proteins URMs/SAMPs (Ubiquitin Related Modifier/Small Archaeal Modifier Protein) have previously been identified. URMs are thought to have evolved from a common antecedent to eukaryotic ubiquitin and likely represent an evolutionary 'missing link' in the adaption of sulphur transfer proteins for covalent modifications. There has been speculation that Urm1 may play a similar role to ubiquitin in the proteasome degradation pathway and we have recently provided evidence to corroborate this. Here the potential for modification of Mre11/Rad50/HerA/NurA by Urm1 was investigated. Indeed Rad50 shows evidence of clear urmylation both in vivo and in vitro. Western blotting and mass spec analysis confirmed the covalent attachment of Urm1 to Rad50. Furthermore I present preliminary evidence that this urmylation can lead to the destruction of Rad50 via a direct physical interaction with the proteasome. This is the first evidence of such a regulatory system for Rad50. Investigating the urmylation and destruction of Rad50 was closely linked to investigating the archaeal proteasome, a close homologue of the eukaryotic proteasome. To date the majority of archaeal core proteasomes examined were believed to consist of only two subunits; alpha and beta. The subunits are arranged into heptameric rings, which then form an alpha/beta/beta/alpha stack with a single channel running through the centre of all four rings. Here we reveal that in Sulfolobus species the inner catalytic chambers are made up of mixed beta rings composed of two subunits. The first plays a crucial structural role but appears catalytically inert, while the second conveys catalytic activity. Here we investigate an inactive complex, containing only the structural beta subunit, and an active complex, containing both beta subunits. First, electron microscopy was performed on both complexes revealing the expected four-layered toroidal stack. Both complexes were subsequently investigated crystallographically. A 3.8 Å structure was determined for the inactive complex. As well as being one of the few archaeal core proteasome structures, this is also an important first step towards structurally investigating the novel three-subunit proteasome. The discovery of active and inactive beta subunits in the archaea brings them even closer to eukaryotic proteasomal systems, making the archaea even more valuable as model systems.

De nombreuses études ont mis en évidence l’importance du contrôle qualité des protéines, c’est à dire des mécanismes de reconformation (chaperons moléculaires) et de dégradation (autophagie, proteasome) des protéines dans différentes pathologies musculaires telles que la dystrophie musculaire d’Ullrich (UCMD), de Duchenne (DMD) ou d’Emery-Dreifuss (EDMD) ; cependant, à l’heure actuelle, aucune n’a été menée sur l’ensemble de ces mécanismes dans un seul et même modèle et sur des cellules musculaires avant leur différenciation en muscles. Nous nous sommes donc intéressés à la fonctionnalité des mécanismes de Contrôle Qualité des Protéines et à leurs interconnexions dans des myoblastes immortalisés de donneurs sains ou de patients atteints de DMD. Nous avons observé une augmentation de l’agrégation protéique dans les cellules DMD. Ce phénomène s’accompagne d’une dérégulation des mécanismes de séquestration par les chaperons moléculaires, conséquence d’une modulation de l’expression des protéines HSPB5 et HSPB8. Les mécanismes de dégradation sont également dérégulés; en effet, nous avons observé d’une part, une diminution de l’activité enzymatique du protéasome ainsi que des molécules d’adressage des protéines multiubiquitinées au protéasome et d’autre part, une augmentation de l’activité du facteur de transcription NF?B, de l’expression de protéines intervenant dans l’autophagie et des complexes BAG3/HspB8 conduisant à une augmentation du flux autophagique. L’ensemble de ces dérégulations reflète l’existence d’un stress d’agrégation protéique dans les myoblastes issus de patients DMD. Dans ce contexte, la modulation pharmacologique du PQC dans ces cellules pourrait représenter une nouvelle stratégie thérapeutique pour la Dystrophie Musculaire de Duchenne / Various studies have highlighted the importance of Protein Quality Control (PQC), including protein refolding (molecular chaperones) and degradation (autophagy, proteasome) mechanisms in inherited muscle disorders such as Ullrich Congenital Muscular Dystrophy (UCMD), Duchenne Muscular Dystrophy (DMD) or Emery-Dreifuss Muscular Dystrophy (EDMD); however, to date, no extensive study has been conducted on these mechanisms in a same model, in muscle cells before muscle differentiation. Thus, we were interested in PQC mechanisms functionality and their interconnection in human immortalized myoblasts from healthy donors or patients suffering from DMD. We observed an increase of protein aggregation in DMD cells. This phenomenon is accompanied by a deregulation of sequestration mechanisms by molecular chaperones, reflected by the modulation of HSPB5 and HSPB8 expression. Degradation mechanisms are also deregulated; indeed, we observed on one hand a decrease of proteasome enzymatic activity and multiubiquitinated proteins UPS-adressing molecules and on the other hand, an increase of NF?B transcription factor’s activity, involved in autophagy, and of BAG3/HSPB8 complexes, leading to an increase of the autophagic flux. These PQC defects reflect the existence of a protein aggregation stress in myoblasts coming from DMD patients. In this context, pharmacological modulation of PQC in these cells could represent a new therapeutic strategy for Duchenne Muscular Dystrophy

O proteassoma 26S é uma estrutura em forma de barril com um núcleo catalítico 20S que é flanqueado por tampas 19S em ambos os lados. Nosso grupo tem demonstrado que o proteassoma 26S é crítico para o desenvolvimento e sobrevivência do Schistosoma mansoni, sendo que mais de 95% dos casais de vermes tratados com o inibidor de proteassoma MG132 apresentaram alteração na postura dos ovos e viabilidade. O tampão 19S é o complexo regulador e funciona no desdobramento e na desubiquitinação das proteínas, antes de sua entrada no complexo 20S, usando enzimas desubiquitinadoras (DUBS). Recentemente, foi demonstrado que a inibição das DUBS, UCHL5 e USP14, que estão reversivelmente ligadas à partícula 19S do complexo 26S, chamada b-AP15, resulta em autofagia, seguida de morte celular, pois leva uma variedade de células de mamíferos ao estresse oxidativo. O objetivo geral deste trabalho foi avaliar o efeito da droga sobre o desenvolvimento reprodutivo de S. mansoni e como possível alvo terapêutico, tendo como objetivos específicos determinar os seus efeitos: na inibição da oviposição in vitro de casais de parasitas Schistosoma mansoni através da contagem dos ovos e expressão do gene p14; avaliar se os órgãos reprodutores dos parasitas sofreram alguma modificação estrutural; se a substância utilizada leva a autofagia; se o tratamento dos casais de parasita com a droga leva a apoptose, ocasionada pela ativação de caspase-3. Este estudo é o primeiro a documentar o papel da droga b-AP15 como um agente esquistossomicida, pois desencadeia alterações ultraestruturais em casais de vermes de S. mansoni. Os seguintes métodos foram utilizados para analisar as alterações: Microscopia Eletrônica de Transmissão (MET), Varredura (MEV) e Confocal; ensaio quantitativo colorimétrico baseado no brometo de 3- (4,5-dimetiltiazol-2-il) -2,5-difenil tetrazólio (MTT), atividade do proteassoma através do substrato Suc-Leu-Le-Al-Tyr-AMC específico para atividade de quimotripsina-like; western blotting; Reação de Polimerase em Cadeia em tempo real e TUNEL. Foram testadas várias concentrações do fármaco (0,2; 0,4; 0,8; 1,6; 3,2 até 50 µM), sendo que a partir de 1,6 µM de b-AP15 ocorreu a inibição da produção de ovos dos casais de parasitas tratados in vitro por 24 h, não havendo alteração da viabilidade, mas mostrou alterações a partir da dosagem de 3,2 µM. A partir de 1,6 µM ocorreram alterações celulares e tegumentares e vermes adultos tratados com 50 µM estavam mortos. O Western blotting mostrou acúmulo de proteínas poliubiquitinadas de alto peso molecular na presença de 1,6µM, havendo mudanças na atividade quimotripsina-like do proteassoma 20S. Os resultados mostraram que o b-AP15 altera a oviposição, a viabilidade e leva à morte de casais de parasitas, reforçando a hipótese de que o sistema ubiquitina/ proteassoma e que as enzimas deubiquitinadoras ligadas a partícula 19S são essenciais para a biologia de S. mansoni. / The 26S proteasome is a barrel structure with a catalytic core 20S that is flanked by 19S caps on both sides. Our group has been showed that 26S proteasomes are critical for Schistosoma mansoni development and survival, being that more than 95% of worms pairs treated with the proteasome inhibitor MG132 showed alteration on egg laying and viability. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14, which are reversibly bound to the 19S particle of the 26S complex, called b-AP15 , results in cell death because it leads to autophagy, followed by cell death in a variety of mammalian cells. The general objective of this work was to evaluate the effect of the drug on the reproductive development of S. mansoni and as a possible therapeutic target. The specific objectives to determine its effects: in inhibiting the in vitro oviposition of couples of Schistosoma mansoni parasites by counting eggs and p14 gene expression; evaluate whether the reproductive organs of the parasites have undergone some structural modification; if the substance used leads to autophagy; if the treatment of the parasite couples with the drug leads to apoptosis, caused by the activation of caspase-3. This study is the first to document the role of the b-AP15 drug as a schistosomicidal agent, as it triggers ultrastructural changes in couples of S. mansoni worms. The following methods were used to analyze the changes: Transmission Electron Microscopy (MET), Scanning (SEM) and Confocal; Colorimetric assay based on 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), proteasome activity through Suc-Leu-Le-Al-Tyr-AMC substrate specific for chymotrypsin-like activity; western blotting; Polymerase chain reaction in real time and TUNEL. Various concentrations of the drug (0.2, 0.4, 0.8, 1.6, 3.2 to 50 ?M) were tested. 1.6 ?M b-AP15 occurred the inhibition of egg production of couples of parasites treated in vitro for 24 h, with no change in viability, but showed changes from the dosage of 3.2 ?M. Cell and tegumentary changes occurred in 1.6 ?M and adult worms treated with 50 ?M were dead. Western blotting showed accumulation of high molecular weight polyubiquitinated proteins in the presence of 1.6?M, with changes in the chymotrypsin-like activity of the 20S proteasome. The results showed that b-AP15 alters the oviposition, viability and leads to the death of couples of parasites, reinforcing the hypothesis that the ubiquitin / proteasome system and the deubiquitinating enzymes bound to the 19S particle are essential for the biology of S. mansoni.

On estime que 10%-20% de la population mondiale est infectée chaque année par des virus influenza A (IAV) saisonniers, causant 250 à 500 000 morts. De plus ces virus présentent des risques de pandémie, et sont à ce titre un problème de santé publique majeur. Le cycle viral est dépendant de la capacité du virus à manipuler le protéome cellulaire. Par ailleurs, le système ubiquitine-protéasome (SUP) cellulaire est impliqué dans de nombreux processus de régulation cellulaires par l'induction de la dégradation de protéines, ou par la modification de leur activation ou de leur localisation sub-cellulaire. Le SUP est une cible privilégiée des virus lors de l'infection. Des études récentes indiquent qu'un réseau d'interactions entre les protéines virales des IAV et les protéines du SUP pourrait contribuer à la réplication virale et l’échappement du virus face au système immunitaire. Cependant ces interactions restent encore mal connues. Nous avons construit une banque contenant 570 facteurs du SUP, ce qui représente environ 60% des facteurs SUP humains connus. Puis nous avons mis au point une méthodologie permettant de réaliser un crible comparatif des interactions entre cette banque SUP et cinq PB2 provenant de souches de virus influenza A de virulence différentes chez l’homme : deux souches saisonnières circulant actuellement dans la population humaine (H1N1pdm09 et H3N2), deux souches hautement pathogènes chez l’homme (H7N9 et H1N1-1918) et une souche de laboratoire (H1N1-WSN). Cette première phase de cartographie a permis de sélectionner 42 facteurs du SUP interagissant avec au moins une des protéines PB2 étudiées. Par ailleurs, l’analyse des similarités de profils d’interaction PB2/UPS des souches étudiées a permis de mettre en évidence une corrélation avec le temps de circulation de chaque souche dans la population humaine. Nous avons ensuite caractérisé le rôle fonctionnel des partenaires de PB2 dans le cycle viral par des expériences de déplétion transitoire de l’expression des facteurs cellulaires par siARN, et validé 36 des 42 facteurs testés. La très grande quantité de facteurs identifiés impliqués dans le cycle viral démontre la qualité de la méthodologie développée pour l’identification de ces interacteurs. Parmi ces facteurs, nous avons étudié plus en détail le rôle de trois deubiquitinases (DUBs) dans l’infection. Nous avons montré que les DUBs sont impliquées dans les phases précoces et tardives du cycle viral. De plus, avec des collègues de Hong Kong nous avons mis en évidence que la DUB OTUB1 est impliquée dans la réponse cellulaire à l’infection produisant des cytokines, et probablement dans l’assemblage des nouveaux virions. Nous avons identifié que la DUB OTUD6A est également impliquée dans les phases tardives du cycle viral. A l’inverse PAN2 qui fait partie des complexes de poly-d’adénylation est impliqué dans les phases précoces. Nous poursuivons nos études afin d’élucider le rôle de ces DUBs dans l’infection par IAV. / An estimated 10%-20% of the world's population is affected each year by seasonal epidemic influenza, causing about 250,000 to 500,000 fatal cases. The pandemic risk reinforces the trait of influenza A virus (IAV) infection as a public health issue. The virus life cycle critically relies on its ability to manipulate the host proteome. Besides, the ubiquitin-proteasome system (UPS) is involved in many regulatory processes in mammalian cells by inducing protein degradation, mediating protein activation or shaping their sub-cellular localisation. Therefore, UPS is a prime target hijacked by viruses. Recent evidence indicates that an intricate regulatory network involving viral proteins and the cellular UPS is likely to contribute to viral replication and immune evasion of influenza A viruses. However, usurpation of the host UPS by IAV is far from being comprehensively deciphered. To gain better understanding, we assessed the interplay between the human UPS and the PB2 subunit of the influenza A virus polymerase through a global proteomic profiling approach. For that purpose, an UPS-dedicated library of 590 human cDNAs, comprising 63% of the whole human UPS, was constituted and characterised. In an initial screen, UPS factors were challenged using a high-throughput split luciferase assay for interaction with the PB2 protein from 5 influenza A strains of different pathogenicity in human. A total of 80 UPS factors emerged as potential PB2 partners, of which 42 were validated as high-confidence PB2 partners for at least one of the strains. Further comparison of interaction profiles of the 5 PB2 with the UPS by hierarchical clustering revealed an interaction dendrogram fitting with the circulation time in the human population.Functional importance of interactors was tested by siRNA-mediated knock down experiments using luciferase tagged recombinant IAV viruses. Depletion of 36 out of the 42 tested UPS factors showed an effect on the infection with all or a subset of IAV strains, underlying the strong functional output of the developed methodology. Among these factors three deubiquitinases (DUBs) were further studied to decipher their involvement in IAV viral cycle. We have shown that they are involved in early and late stage of the infection and began to draw their function in viral cycle. We demonstrated with our colleagues in Hong-Kong that OTUB1 is involved in the host cytokine response and most probably in virus assembly. OTUD6A was also shown to be implicated in late stages of the infection but we still don't know its exact role. Contrariwise, the inactive DUB PAN2, which is part of poly-deadenylation complexes, is implicated in early phase of IAV infection, but surprisingly apparently not through viral mRNA regulation. More work is on-going to precise by which mechanisms these DUBs are implicated in IAV infection.

ABSTRACT PURIFICATION AND CHARACTERIZATION OF CYTOPLASMIC AND PROTEASOME ASSOCIATED CHYMOTRYPSIN-LIKE PROTEASES FROM THERMOPLASMA VOLCANIUM &Ouml / zdemir, F.inci Ph.D., Department of Biology Supervisor: Prof. Dr. Semra Kocabiyik September, 147 pages In this study, two novel cytoplasmic serine proteases were isolated and characterized from thermophilic archaea Thermoplasma volcanium. The first protease was purified by ion exchange and affinity chromatographies and identified as a chymotrypsin-like serine protease mainly based on its substrate profile and inhibition pattern. The presence of protease activity was analyzed by gelatin zymography which was detected as a single band (35 kDa). Optimum temperature was found to be 60oC for azocasein hydrolysis and 50oC for N-Suc-Phe-pNA hydrolysis. Optimum activity was observed in the pH range of 6.0-8.0 with a maximum value at pH 7.0. The Km and Vmax values for the purified protease were calculated to be 2.2 mM and 40 &micro / moles of p-nitroanilide released min-1.ml-1, respectively, for N-Suc-Phe-PNA as substrate. Ca2+ and Mg2+ at 4 mM concentrations were the most effective divalent cations in activating the enzyme. In the second stage of this study, 20S proteasome of Tp. volcanium with substantial chymotrypsin-like activity was purified and characterized. This enzyme complex was purified with 19.1 U/mg specific activities from cell free extract by a four-step procedure. SDS-PAGE analysis revealed two strong bands with relative molecular masses of 26 kDa (&amp / #945 / -subunit) and 21.9 kDa (&amp / #946 / -subunit). Tp. volcanium 20S proteasome predominantly catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residue Phe (chymotrypsin-like activity) in short chromogenic peptides. Low-level hydrolyzing activity was also detected carboxyl to basic residue Arg (trypsin-like activity). Chymotrypsin-like activity of Tp. volcanium 20S proteasome was significantly inhibited by chymotrypsin specific serine protease inhibitor chymostatin. When N-CBZ-Arg was used which is a substrate for trypsin, 20S proteasome was strongly inhibited by TLCK. The optimum temperature for Ala-Ala-Phe-pNA hydrolysis by the Tp. volcanium 20S proteasome was 55oC and the optimum pH was 7.5. The chymotryptic activity was significantly enhanced by divalent cations such as Ca+2 and Mg2+ at high concentrations, i.e. 125-250 mM. Keywords:Serine protease, 20S proteasome, archaea, thermophilic protease, Thermoplasma volcanium, chymotrypsin-like serine protease.

Targeted protein degradation plays a central role in eukaryotic cell regulation and homeostasis. The ubiquitin proteasome system (UPS) is involved in a large number of cellular events, being, day by day, more difficult to find pathways without links with the system. Proteins are marked for degradation by ubiquitin ligases that append polyubiquitin signals, which can recruit the targeted protein to the 26S proteasome for degradation. In the process, polyubiquitin chains appended to the target protein are disassembled into free monoubiquitin for subsequent rounds of substrate tagging by means of deubiquitinating enzymes. Rpn10 is a proteasome receptor that recognizes the Lys48-linked polyubiquitin degradation signal by means of a ubiquitin-interacting motif (UIM). Mutation of the UIM domain of Rpn10 significantly ablates the proteolytic capacity of the proteasome. Rpn10 also contains a Von Willebrand factor A (VWA) domain which is responsible of Rpn10 interactions inside the proteasome. Rpn10 is in equilibrium with a pool of free protein so that it functions as both, a receptor at the proteasome, and an extraproteasomal adaptor. In the present work, we have shown that Rpn10 is monoubiquitinated (Rpn10-mUb) in vivo and that Rpn10-mUb is found in both proteasomal and non-proteasomal pools. Levels of Rpn10-mUb are regulated in vivo by Rps5, a NEDD4 ubiquitin-ligase protein family, and Ubp2, a deubiquitinating enzyme. Our observations link for first time monoubiquitin signal with proteasome regulation. Monoubiquitination strongly inhibits Rpn10 to interact with ubiquitin conjugates by a specific interaction in cis between Rpn10 UIM domain and the linked monoubiquitin. By means of genetic and proteomics tools we found that four sites of Rpn10 sequence are subjected to monoubiquitination by Rsp5 (Lys71, Lys84, Lys99 and Lys268), but Lys84 within its VWA domain is the preferred one. Ubiquitination of Rpn10 inhibits its ability to bind polyubiquitinated substrates, thus functioning as mechanism of inactivation of this receptor. Interestingly, our findings suggest that Rpn10 monoubiquitination could decrease proteasome activity. The UIM of Rpn10 also strongly interacts with the ubiquitin-like domain (Ubl) of Dsk2, a polyubiquitin-binding protein. Furthermore, extraproteasomal Rpn10 plays a critical role in filtering Dsk2 and its substrates from the proteasome. We evaluated the affinity of several forms of Rpn10-mUb to Dsk2 and found that Ub-UIM interaction in cis of Rpn10-mUb impairs the binding of Dsk2 in trans. These results suggest that in an extraproteasomal context, Rpn10 monoubiquitination could regulate the Dsk2-Rpn10 interaction. Notably, the proteasomal pool of Rpn10-mUb is suppressed under perturbations that promote the proteolytic pathway, such as stress by temperature and oxidative stress, whereas the extraproteasomal pool remains fairly constant. To assess whether the distinct behavior of the Rpn10-mUb pools correlated with the modification of different lysine residues of Rpn10, we set up assays of absolute quantification of the two major ubiquitination sites, Lys84 and Lys268, by mass spectrometry (AQUA). The obtained results clearly indicate that Lys84 is the main target of Rsp5 ligase in both pools of Rpn10, enhancing the role of the VWA domain in Rpn10 ubiquitination. Finally, we have combined ubiquitin remnant profiling with quantitative proteomic approaches to identify ubiquitinated species that are increased in RPN10-RAD23 deletion, under standard growth conditions and cold-shock stress. Enriched conjugates have been distributed across different biological functions being proteins involved in RNA processing and transport metabolism accounting for the biggest fractions. Further molecular biology approaches and informatic analysis are required to address which proteins are likely to be true UPS substrates as opposed to proteins regulated by ubiquitination in a non-proteolytic manner. Overall, our results provide a novel evidence of involvement of monoubiquitin signals in the regulation of the availability of substrates to bind proteasomal surfaces. Considering that substrate binding is the first event in the multicatalytic process promoted by the proteasome, the control of the accessibility of proteasome receptors is a crucial level of proteasome regulation. Thus, if the NEDD4 enzyme catalyzed monoubiquitination of Rpn10 inhibits proteasome activity, the control of this reaction could be an interesting target. Classical proteasome inhibitors, such as Bortezomib, which target the proteolytic activity of the core particle of the proteasome, are used as anticancer drugs. We propose that Rpn10 monoubiquitination could be considered as a new target in cancer research. / La degradació de proteïnes juga un rol essencial en la regulació i homeostasi de les cèl•lules eucariotes. Les proteïnes són marcades per a la seva degradació per ubicuitina lligases les quals annexen cadenes de poliubicuitina enviant de manera específica la proteïna al 26S proteasoma on serà degradada. Rpn10 és un receptor del proteasoma que reconeix cadenes d’ubicuitina a través del seu motiu UIM (ubiquitin-interacting motif). Rpn10 també conté un domini VWA el qual és responsable de les interaccions de Rpn10 en el seu context proteasomal. Rpn10 també es troba en equilibri amb una fracció no proteasomal. En el present treball hem demostrat que Rpn10 està monoubicuitinat (Rpn10-mUb) in vivo i que aquesta modificació es troba en ambdues fraccions proteasomal i extraproteasomal. Els nivells de Rpn10-mUb estan regulats in vivo per Rsp5, una ubicuitina lligasa de la família de les NEDD4, i Ubp2, una deubicuitinasa. Mitjançant mètodes genètics i de proteòmica es van trobar quatre lisines modificades per ubicuitina en la seqüència de Rpn10 (Lys71, Lys84, Lys99 and Lys268) éssent la Lys84 la diana preferent de Rsp5. La monoubicuitinació inhibeix la capacitat de Rpn10 d’unir substrats poliubicuitinats i en, conseqüència, és un mecanisme d’innactivació d’aquest receptor. També s’ha trobat que Rpn10-mUb disminuiex l’activitat del proteasoma. Els nostres resultats relacionen per primera vegada la monoubicuitinació amb la regulació del proteasoma, procés que imita la droga Bortezomib. Es proposa que Rpn10-mUb podria considerar-se com a nova diana en la teràpia contra el càncer.

Raman spectroscopy offers much better spectral selectivity but its usage has been limited by its poor sensitivity. The discovery of surface-enhanced Raman scattering (SERS) effect, which results in increased sensitivities of up to 108-fold for some compounds, has eliminated this drawback. A new SERS active substrate was developed in this study. Silver nanoparticle-doped polyvinyl alcohol (PVA) coated SERS substrate prepared through chemical and electrochemical reduction of silver particles dispersed in the polymer matrix. Performances of the substrates were evaluated with some biologically important compounds. The specific detection of DNA has gained significance in recent years since increasingly DNA sequences of different organisms are being assigned. Such sequence knowledge can be employed for identification of the genes of microorganisms or diseases. In this study, specific proteasome gene sequences were detected both label free spectrophotometric detection and SERS detection. In label free spectrophotometic detection, proteasome gene probe and complementary target gene sequence were attached to the gold nanoparticles separately. Then, the target and probe oligonucleotide-modified gold solutions were mixed for hybridization and the shift in the surface plasmon absorption band of gold nanoparticles were followed. SERS detection of specific nucleic acid sequences are mainly based on hybridization of DNA targets to complementary probe sequences, which are labelled with SERS active dyes. In this study, to show correlation between circulating proteasome levels and disease state we suggest a Raman spectroscopic technique that uses SERGen probes. This novel approach deals with specific detection of elevated or decreased levels of proteasome genes&rsquo / transcription in patients as an alternative to available enzyme activity measurement methods. First, SERGen probes were prepared using SERS active labels and specific proteasome gene sequences. Then DNA targets to complementary SERGen probe sequences were hybridized and SERS active label peak was followed.

Aryl hydrocarbon receptor (AHR) is a sensor protein, activated by aromatic chemical species for transcriptionally regulating xenobiotic metabolizing enzymes. AHR is also known to be involved in a variety of pathogenesis such as cancer, diabetes mellitus, cirrhosis, asthma, etc. The AHR signaling induced by xenobiotics has been intensively studied whereas its physiological role in the absence of xenobiotics is poorly understood. Despite a number of ligands of AHR have been reported thus far, further applications are still hampered by the lack of specificity and/or the partially agonistic activity. Thus, a pure AHR antagonist is needed for deciphering the AHR cryptic as well as potential therapeutic agent. The Proteolysis Targeting Chimera (PROTAC) is a bi-functional small molecule containing a ligand and proteolysis inducer. PROTAC recruits the target protein to proteolysis machinery and elicits proteolysis. Thus far, a number of PROTAC have been prepared and demonstrated to effectively induce the degradation of targeted protein in cultured cells, validating PROTAC as a useful research tool. In the present study, PROTACs based on apigenin was prepared and demonstrated to induce the degradation of AHR, providing the proof of concept. To improve activity, a synthetic structure, CH-223191, was optimized for antagonistic activity by positional scanning identifying several AHR antagonists. PROTACs based on the optimal structure were prepared and assessed their biological activity. The products and synthetic scheme described hereby will be helpful for the further understanding on AHR biology as well as for developing therapeutic agents targeting AHR.

Monogenic disorders, i.e., disorders caused by mutations in a single gene, are rare and clinically heterogeneous conditions. Identification of the genetic cause of monogenic traits can bring new insights into molecular pathways and disease mechanisms. The aims of the present study were to identify the mutant genes in two autosomal recessive skin disorders and to characterize the functions of the mutated genes.  In order to identify candidate genes for the two disorders whole-genome SNP analysis, homozygosity mapping and gene sequencing were used. Autosomal recessive congenital ichthyosis (ARCI) is a group of disorders characterized by extensive scaling and redness of the skin.  A subgroup of ARCI patients (n=27) was selected based on specific ultrastructural aberrations in their skin, revealed by electron microscopy. Mutations were identified in the Ichthyin gene in 93% of the selected patients, indicating a strong association between mutant Ichthyin and the specific morphological abnormalities. Ichthyin mRNA levels were shown to increase during keratinocyte differentiation in cells from healthy and affected individuals. Electron microscopy revealed a localization of ichthyin protein to keratins and desmosomes in epidermis. Staining of epidermal lipids identified aberrant lipid aggregates in skin sections of patients with Ichthyin mutations, indicating a role for Ichthyin in epidermal lipid metabolism. In twelve KLICK syndrome patients with ichthyosis, palmoplantar keratoderma and keratotic striae on joints, a single-nucleotide deletion was identified in the 5’ region of the proteasome maturation protein (POMP) gene.  The deletion caused an increase in the proportion of POMP transcripts with long 5’ UTR’s in patient keratinocytes.  Immunohistochemical analysis of differentiated skin cell layers revealed aberrant expression of POMP, proteasome subunits and the skin protein filaggrin in patients. CHOP expression, associated with endoplasmic reticulum stress, was increased in the same layers. siRNA silencing of POMP in cell cultures reduced proteasome subunit levels and induced expression of CHOP.  The results indicate that the mutation in KLICK patients causes POMP and proteasome insufficiency with subsequent cellular stress. This study conclusively contributes to the understanding of epidermal physiology and the pathogenesis of two inherited skin diseases.