Post-translational modifications are chemical changes that occur to proteins after their synthesis, which are essential to their function and regulation. Ubiquitination is a post translation modification that serves key roles in the regulation of proteins. USP7 is a deubiquitinase that has several critical substrates important for human health and disease, including the cancer relevant proteins PTEN, p53, MDM2 and DMNT1. Most of these substrates have been identified by cell biology or proteomics experiments, but a detailed biochemical and structural analysis is lacking, likely due to the challenge of generating site-specific and stoichiometrically ubiquitinated proteins. Therefore, we leveraged our expertise in protein semi-synthesis to generate these ubiquitinated substrates to study USP7’s ability to recognize and hydrolyze the ubiquitin, which will reveal key details on how USP7 selects its substrates. In our investigation, we generated several mono-ubiquitinated peptides, that could be functionalized later to install on the protein, and assayed USP7’s ability to hydrolyze the ubiquitin. / 2026-03-06T00:00:00Z

Câncer se destaca no contexto de patologias por ser uma das doenças que mais acometem mortes por ano, sendo caracterizada como um conjunto de doenças multifatoriais que tem em comum o crescimento desordenado de células que invadem tecidos e órgãos, podendo espalhar-se para outras regiões do corpo, dando origem às metástases. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem carácter invasivo e migratório, além de se tornarem mais resistentes às drogas. Durante este processo, ocorrem inúmeras alterações celulares que modificam a estabilidade proteica e/ou promovem sua translocação subcelular, o transporte de proteínas para a membrana, alterações no citoesqueleto e incluindo o envio de proteínas para degradação pelo proteassoma. A desregulação de fatores de transcrição e modificação pós traducional de proteínas são fatores que podem levar à EMT. Após a eficiente indução da EMT in vitro utilizando o inibidor de histonas deacetilase (SAHA) em células de adenocarcinoma de mama MCF-7, foram realizadas análises proteômicas envolvimento os inibidores relacionados ao sistema ubiquitina proteassoma, MG132 e P5091. A modulação por inibição de USP7 resultou em variação da expressão de diversas proteínas biomarcadoras da EMT (SNAIL, ?-Catenina, CDK1) e proteínas envolvidas no ciclo celular (P53 e CDK1). O estudo proteômico permitiu a correlação do processo da EMT por SAHA com as vias de modificações pós traducionais relacionadas ao sistema ubiquitina proteassoma, e ainda propõe USP7 como alvo de estudos detalhados para EMT com potencial proposta terapêutica / Cancer stands out in the context of pathologies because it is one of the diseases that most affect deaths per year, being characterized as a set of multifactorial diseases that has in common the disordered growth of cells that invade tissues and organs, being able to spread to other regions of the body, giving rise to metastases. An important step in the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of the epithelial phenotype and acquisition of the mesenchymal phenotype by the tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. During this process, numerous cellular alterations occur that modify the protein stability and/or promote its subcellular translocation, the transport of proteins to the membrane, changes in the cytoskeleton and including the sending of proteins for degradation by the proteasome. Deregulation of transcription factors and posttranslational modification of proteins are factors that can lead to EMT. After an efficient induction of EMT using the histone deacetylase inhibitor (SAHA) in MCF-7 breast adenocarcinoma cells, proteomic analyzes were performed involving inhibitors related to the ubiquitin proteasome system, MG132 and P5091. Modulation by inhibition of USP7 resulted in varying expression of various EMT biomarker proteins (SNAIL, ?-Catenina, CDK1) and cell cycle (P53 e CDK1). The proteomic study allowed the correlation of the SAHA EMT process with the posttranslational modifications pathways related to the ubiquitin proteasome system and also proposes USP7 as the target of detailed studies for EMT with potential therapeutic proposal

Multiple Myeloma (MM) is an incurable plasma cell malignancy and, although novel treatment regimes in the past decade have improved patient outcome, long-term treatment leads to relapse and refractory disease. The centrosomal kinase NEK2 is found overexpressed in MM and promotes chromosomal instability, drug resistance and increased proliferation. Although much research shows NEK2 having a detrimental effect in cancer, much of its mechanisms of overexpression and drug resistance has not been studied in detail. In this work we expand our understanding of NEK2 in MM. Using Tandem Affinity Purification coupled with Mass Spectrometry, we show that NEK2 directly interacts with the de-ubiquitinase USP7. We confirm this interaction in cell lines of MM and lung cancer. Since USP7 has been shown to have important cancer-promoting roles we tested if USP7 was necessary for NEK2-driven bortezomib resistance. We found that USP7 shRNA was sufficient to sensitize the bortezomib resistant NEK2 overexpressing cells to bortezomib. Surprisingly, we found that USP7 inhibition with shRNA or by treatment with the small molecule USP7 inhibitor P5091 led to depletion of NEK2 protein in every cell line tested. Previous research shows USP7’s main function is a de-ubiquitinase and, since NEK2 is a target of the ubiquitin-proteasome system, we hypothesized USP7 may be de-ubiquitinating NEK2. Through western blots and immunoprecipitations, we show the NEK2-USP7 interaction promotes the de-ubiquitination and subsequent stabilization of NEK2, presenting USP7 as the first discovered de-ubiquitinating enzyme of NEK2. To understand how NEK2 promotes drug resistance in cancer we studied a previously published list of NEK2-regulated genes and, using the UCSC genome browser (Track Name:GM12878+TNFa RELA) ChIP-seq data, we found approximately half of these genes have the NF-κB transcription factor p65 bound throughout the gene sequence. We also produced a signaling score using an average of 11 known targets of NF-κB and patients with high NEK2 showed a significantly increased score of NF-κB signaling. Additionally, through western blots and immunofluorescence, we found that patients with high NEK2 protein levels consisitently had activation higher signal of p65 protein and phosphorylated p65 at Serine 536, indicative of increased activity. We then causally show NEK2 activates canonical NF-κB by performing western blots and a dual-luciferase reporter assay on control and NEK2 overexpressing cells. Using AKT and PP1α inhibitors, we found that NEK2 drives NF-κB by phosphorylating and inactivating PP1α, leading to hyperactive AKT. Using this model of NEK2-NF-κB activation, we aimed at targeting NEK2 directly with the small molecule drugs INH1 (depletes NEK2 protein) and P5091 (inhibits USP7 activity) in empty vector control cells, NEK2 overexpressing cells or cells with an acquired drug resistance phenotype. Our results show that both INH1 and P5091 can overcome bortezomib resistance in cell lines and in vivo. Another aspect of MM disease we targeted in this work was bone disease. Bone disease in MM is common and causes bone pain and fractures but a much is still regarding what drives these lesions. We found that NEK2 expression in patients correlates with a presence of bone lesions, based on FDG-PET scan and MRI. Using our previously published list of NEK2 regulated genes, we found Heparanase (HPSE) is directly correlated to NEK2 expression. HPSE is an extracellular protein shown to promote differentiation of the bone destroying cell, osteoclast. Using western blots, RT-qPCR and ELISA, we found NEK2 increases HPSE expression and extracellular release. HPSE was also on the list of genes upregulated by NEK2 found to have p65 bound to the gene, thus we tested if NEK2 was driving HPSE through the NF-κB. Accordingly, we found NEK2 drives HPSE through the NF-κB pathway and, consistent with our previous results, in a USP7-dependent manner. Using bone marrow macrophages and conditioned media from empty vector control or NEK2 overexpressing cells, we found NEK2 promtoes increased differentiation of osteoclasts and inhibition of HPSE blocked this effect, strongly suggesting HPSE is the mediator of this effects. Importantly these findings were recapitulated in vivo. Empty vector or NEK2 overexpressing cells were injected through the tail vein to allow dissemination to the bone marrow. microCT and Xray revealed mice injected with NEK2 overexpressing cells showed reduced bone density, compared to empty vector cells. Additionally, H&E and TRAP staining confirmed our in vitro results by showing higher osteoclast levels in bone sections of mice injected with NEK2 overexpressing cells. Lastly, we show a novel role for the ATPase TRIP13 as a cofactor for USP7 de-ubiquitinating activity. TRIP13 is overexpressed in cancer, has been shown to be an oncogene and promotes drug resistance. By systematically targeting TRIP13 overexpressing cells with drugs that inhibit different pathways we found TRIP13 drug resistance is diminished by inhibiting USP7. We found that TRIP13 binds with USP7 and by western blots and immunoprecipitations we show it is necessary for the de-ubiquitination of NEK2. Furthermore, we also found TRIP13 shows a hyperactive USP7 phenotype, shuttling PTEN out of the nucleus and stabilizing MDM2, in a USP7 dependent manner. In summary, this work shows the de-ubiquitinase USP7, coupled with the ATPase TRIP13 stabilizes NEK2 by de-ubiquitination, this leads to accumulation of NEK2 and activation of the canonical NF-κB pathway through PP1α/AKT, which promotes drug resistance and activates HPSE, increasing osteoclast differentiation and bone destruction.

Die Akkumulation fehlerhafter Proteine im Endoplasmatischen Retikulum (ER) induziert den „unfolded protein response“ (UPR) - Signalweg zur Überwindung dieser zellulären Stress-Situation. Ein in Säugern stark UPR-induziertes Gen kodiert für das Ubiquitin-Domäne-Protein Herp. Herp interagiert mit der E3-Ligase Synoviolin, einer zentralen Komponente von Multiproteinkomplexen, welche die ER assoziierte Protein-Degradation (ERAD) vermitteln. Abhängig von seiner Ubiquitin-ähnlichen (‘UBL’) Domäne wird Herp für den effizienten Abbau von Synoviolin-Substraten benötigt. Der zugrundeliegende molekulare Mechanismus dieser Funktion von Herp ist kaum bekannt. In der vorliegenden Studie wurde gezeigt, dass Herp kontinuierlich an Synoviolin-basierten Komplexen umgesetzt wird, aber kein Substrat ist. Da sowohl Depletion als auch Stabilisierung von Herp zum verminderten Abbau von Synoviolin-Substraten führt, lässt sich schlussfolgern, dass der kontinuierliche Umsatz von Herp entscheidend ist für ERAD. Weiterhin regulierte Herp die Zusammensetzung Synoviolin-basierter Komplexe. Das deubiquitinierende Enzym Usp7 ist über seine Bindung an Herp mit Synoviolin assoziiert. Usp7 beeinflusste aber nicht die Stabilität von Herp oder ERAD-Substraten. Zusätzlich verstärkte Herp die Interaktion zwischen dem CUE-Domäne-Protein AUP1 und Synoviolin. In Abhängigkeit von der CUE-Domäne steigerte AUP1 den ERAD-Prozess. Auch das Herp-Homolog Herp2 war mit Synoviolin-basierten Komplexen assoziiert. Im Gegensatz zu Herp wurde Herp2 nicht durch den UPR-Signalweg induziert, war stabil und interagierte nicht Usp7. Diese Daten unterstreichen die einzigartige Funktion von Herp im ERAD-Prozess. Schlussfolgernd ist Herp eine dynamische ERAD-Komponente, welche die Rekrutierung akzessorischer Proteine an Synoviolin vermittelt und damit die Ubiquitinierung von Synoviolin-Substraten ermöglicht. Diese Daten zeigen die kritische Rolle von Herp für die Beseitigung fehlerhafter Proteine und das Überleben der Zelle. / The accumulation of aberrant proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR) pathway for surmounting this cellular stress situation. One of the strongly UPR-induced genes in mammalia encodes the ubiquitin domain protein Herp. Herp interacts with the E3 ligase Synoviolin, a central component of ER associated protein degradation (ERAD) mediating multiprotein complexes. Dependent on its ubiquitin-like (UBL) domain, Herp is required for the efficient degradation of Synoviolin substrates. The molecular mechanism underlying this function of Herp is poorly understood. In the present study, it was shown that Herp is continuously exchanged at Synoviolin based complexes. However, Herp did not serve as a Synoviolin substrate. Since both stabilisation and depletion of Herp resulted in the impaired degradation of Synoviolin substrates, the continuous turnover of Herp seems to be decisive for ERAD. Herp was also shown to regulate the composition of Synoviolin based complexes. The deubiquitinating enzyme Usp7 was linked to Synoviolin via its interaction with Herp. However, Usp7 did not influence the stability of Herp or ERAD substrates. In addition, Herp improved the association of the CUE domain protein AUP1 with Synoviolin. AUP1 triggered the ERAD process in a CUE domain dependent manner. Also Herp2, a homologue of Herp, was found to associate with Synoviolin based complexes. However, in contrast to Herp, Herp2 was not induced by the UPR, was stable, and did not bind Usp7 supporting the idea of Herp having a unique function in ERAD. In conclusion, Herp is a dynamic ERAD component recruiting accessory proteins to Synoviolin thus enabling Synoviolin dependent ubiquitination of substrates. These findings point out the crucial role of Herp for the elimination of misfolded proteins, which is important for cell survival.

Inflammation is the response of the body to injury or threats. Immune cells such as macrophages have a crucial role in controlling and regulating this process. The potent pro-inflammatory cytokines interleukin (IL)-1beta and IL-18 are synthesized by macrophages as inactive precursors which activation follows a unique mechanism involving the activation of caspase-1 by assembly of a macromolecular complex called the inflammasome. However, the assembly of the inflammasome is a double-edged sword. Although inflammasome activation is necessary for a normal inflammatory response, its malfunction can trigger and contribute to inflammatory disorders such as gout, arthritis or cryopirin-associated periodic syndromes (CAPS). The fine regulation of this mechanism and the cell death associated with it is key for the outcome of the inflammatory process. In this thesis we tackle three aspects of the mechanisms and consequences of inflammasome activation. First we studied the role of the deubiquitinases USP7 and USP47 in inflammasome activation. We showed how USP7 and USP47 activity is increased upon danger signals and how that is necessary for the assembly of the inflammasome. We also pointed how their inhibition dampens the deubiquitination of ASC using a BRET2 assay. Second we examined how the activity of IL-18 is controlled by the release of IL-18BP during inflammasome activation. We showed how IL-18BP release increased upon membrane permeabilization and pyroptosis. This release happens in other types of lytic cell (necrosis and necroptosis) death but not in apoptosis. Finally, we showed that this IL-18BP acute release dampens IL-18 signalling and IFN gamma production by PBMCs. These results demonstrate a novel mechanism by which lytic cell death could dampen IL-18-driven inflammation and highlights a key role for IL-18BP in inflammasome related diseases. Finally we studied the role of inflammasome in lung epithelial cells as a model to investigate lung infections. We found that lung epithelial cells lack NLRP3 inflammasome activity and components, but express caspase-4 and caspase-8 which could have a role in the release of IL-1 family of cytokines. To conclude we showed how lung epithelial release IL-18 upon Aspergillus fumigatus infection. Overall, this thesis enhances our understanding of the mechanisms that control IL-1beta and IL-18 activity by regulating inflammasome activation and by understanding the consequences of its activation.

Die ER-assoziierte Proteindegradation (ERAD) ist Teil des Qualitätskontrollsystems am ER, um der Akkumulation von fehlgefalteten Proteinen im ER entgegenzuwirken. Hierbei werden ERAD-Substrate mit Hilfe von E3-Ligasen wie z.B. HRD1 ubiquityliert und anschließend durch den p97-Ufd1-Npl4 Komplex aus der ER-Membran extrahiert. Im Zytosol werden diese extrahierten Proteine vom 26S Proteasom abgebaut. Für die Retrotranslokation von ERAD- Substraten werden zudem die Membranproteine Derlin-1 und VIMP benötigt, welche mit p97 assoziieren und einen Proteinkomplex bilden. HERP ist ein ER-lokalisiertes Protein, dessen Synthese durch den UPR (unfolded protein response) als Antwort auf die Akkumulation von fehlgefalteten Proteinen im ER induziert wird. Dies deutet auf eine Rolle von HERP im ERAD hin. Interessanterweise besitzt HERP eine sogenannte UBL-Domäne. Für andere Proteine mit UBL-Domäne konnte eine Interaktion dieser Domäne mit dem Proteasom nachgewiesen werden. Daher kann angenommen werden, dass HERP ebenfalls mit dem Proteasom interagiert und dies zur ER- Membran rekrutiert, wo es für den Abbau von ERAD-Substraten benötigt wird. Das Ziel der vorliegenden Arbeit war es, die Rolle von HERP innerhalb des UPR zu ermitteln. Die hier präsentierten Daten zeigen, dass HERP essentiell für den Abbau des ERAD-Modell- Substrates CD3-delta ist. Somit hat HERP tatsächlich eine Rolle im ERAD. Außerdem wird eine direkte Interaktion von HERP mit der E3-Ligase HRD1 nachgewiesen. Es wird zudem gezeigt, dass HERP und HRD1 einen Proteinkomplex mit p97, Derlin-1 und eventuell auch mit VIMP bilden. Dieser ERAD Komplex ist folglich sowohl für die Ubiquitylierung als auch die Retrotranslokation von ERAD-Substraten verantwortlich und garantiert somit die effiziente Prozessierung von Proteinen aus dem ER. Zudem wird gezeigt, dass die UBL-Domäne von HERP im Gegensatz zu anderen UBL- Domänen nicht mit dem Proteasom interagiert. Somit kann nicht mehr davon ausgegangen werden, dass Proteasombindung eine Gemeinsamkeit aller Proteine mit UBL-Domäne ist. Dagegen wird eine Interaktion der UBL-Domäne von HERP mit dem deubiquitylierenden Enzym USP7 nachgewiesen. Dies deutet darauf hin, dass auch Deubiquitylierung eine wichtige Rolle im ERAD-Prozess spielt. / ER-associated protein degradation (ERAD) is part of the ER quality control system dealing with the accumulation of misfolded proteins in the ER. This process requires polyubiquitylation of ERAD substrates involving E3 ligases, such as HRD1, and their subsequent extraction from the ER membrane by the p97-Ufd1-Npl4 complex. Retrotranslocation of substrates into the cytosol for degradation by the 26S proteasome also involves the membrane proteins Derlin-1 and VIMP, which are associated with p97 to form a protein complex. The ER-resident protein HERP was shown to be upregulated by the unfolded protein response pathway (UPR) upon the accumulation of misfolded proteins in the ER. It was therefore considered to function in ERAD. Interestingly, HERP contains a UBL domain. In other proteins this domain facilitates an interaction with the proteasome, suggesting that HERP might recruit the proteasome to the ER membrane for efficient ERAD. The aim of this study was to investigate the function of HERP within the UPR. The findings presented here demonstrate that HERP is essential for the degradation of a model ERAD substrate. Thus, HERP indeed has a role in ERAD. Moreover, the data show that HERP directly interacts with the E3 ligase HRD1 and the two proteins form a common protein complex with p97, Derlin-1 and possibly also with VIMP. This suggests that both ubiquitylation and retrotranslocation of ER proteins are performed by one protein complex, enabling an efficient processing of ERAD substrates. This study also demonstrates that the UBL domain of HERP does not share the proteasome binding property of other UBL domains, suggesting that proteasome binding cannot be considered a general feature of all UBL domains. Instead, the HERP UBL domain is able to interact with the deubiquitylating enzyme USP7. Therefore, deubiquitylation might also be an important aspect in the proteasome-dependent degradation of misfolded ER proteins.