Deubiquitinating Enzyme Inhibitors Block Chikungunya Virus Replication.

Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection.

Inhibition of 19S proteasome deubiquitinating activity in Schistosoma mansoni affects viability, oviposition, and structural changes.

The proteasome is the key player in the cellular protein degradation machinery and is pivotal for protein homeostasis and Schistosoma mansoni (S. mansoni) survival. Our group study provides insights into proteasome inhibitors and reveals that selective schistosomiasis agents represent an interesting branch of proteasome research linked to the development of new drugs for this neglected disease. Here, we explored the phenotypic response of S. mansoni to b-AP15, a bis-benzylidine piperidone that inhibits 26S proteasome deubiquitinases (DUBs), ubiquitin-specific protease 14 (USP14), and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5). b-AP15 induces a modest decrease in egg production in vitro and reduces viability, leading to the death of parasite couples. This inhibitor also induces a twofold increase in the accumulation of polyubiquitinated proteins in S. mansoni adult worms and causes tegument changes such as disintegration, wrinkling, and bubble formation, both throughout the length of the parasite and in the oral sucker. b-AP15 alters the cell organelles of adult S. mansoni worms, and we specifically observed mitochondrial alterations, which are suggestive of proteotoxic stress leading to autophagy. Taken together, these results indicate that the deubiquitinase function of the proteasome is essential for the parasite and support the hypothesis that the proteasome constitutes an interesting drug target for the treatment of schistosomiasis.

A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

Preliminary study of the enzyme ubiquitin carboxylterminal hydrolase 14 (UBP6) in Giardia intestinalis: structural bioinformatic analysis and transcriptional profile during encystation / Estudio preliminar de la enzima ubiquitina carboxiterminal hidrolasa 14 (UBP6) en Giardia intestinalis: análisis bioinformático de su estructura y perfil transcripcional durante la enquistación / Estudo preliminar da enzima ubiquitina carboxi-terminal hidrolasa 14 (UBP6) em Giardia intestinalis: análise bioinformática estrutural e perfil transcricional durante encistamento

This paper presents a combined approach with two aims. The first is to analyze the reported sequence of the enzyme ubiquitin carboxyl-terminal hydrolase 14 of Giardia intestinalis (UBP6) through computational methods to find components related with its hypothetical function. The second is to determine if the protein-coding gene is expressed in G. intestinalis and, if such is the case, also determine its transcription pattern along the life cycle of the parasite. It was established that the protein belongs to the family of Cys-dependent deubiquitinases and more specifically to ubiquitin specific proteases (USPs). Moreover, the catalytic center with the complete triad as well as typical features of the USP motif were also identified. Since the computational findings suggest that the enzyme could be functional, reverse transcription coupled to PCR was used as a first approach to establish if in fact the coding gene is expressed in the parasite. Interestingly, it was found not only that the gene is expressed, but also that there is a transcription variation along the life cycle of the parasite. These two findings are the starting point for further studies since they tentatively suggest that this enzyme could be involved in the protein turnover that occurs during parasite encystation. Although preliminary, this study is the first report concerning the study of a specific deubiquitinating enzyme in the parasite G. intestinalis.

En este trabajo se presenta una estrategia combinada que buscaba, primero, analizar por métodos computacionales la secuencia de la enzima ubiquitina carboxilo-terminal hidrolasa 14 de Giardia intestinalis (UBP6) reportada para buscar componentes relacionados con su función hipotética y segundo, determinar si el gen que codifica para la proteína se expresa en G. intestinalis y si lo hace, cómo es su patrón de transcripción a lo largo del ciclo de vida del parásito. Se encontró que la proteína pertenece a la familia de deubiquitinasas dependientes de cisteína y más específicamente a las proteasas específicas para ubiquitina (USPs por ubiquitin specific proteases). También se identificaron el centro catalítico con la triada completa así como características típicas del motivo USP. Teniendo en cuenta que los resultados computacionales sugieren que la enzima puede ser funcional, se usó la técnica de transcripción reversa acoplada a PCR como un primer acercamiento para establecer si el gen codificante se expresa en el parásito. De manera interesante, se determinó no solo que el gen se expresa sino que existe una variación de su transcripción a lo largo del ciclo de vida del parásito. Estos hallazgos son el punto de partida para posteriores estudios ya que sugieren de manera preliminar que esta enzima podría estar involucrada en el recambio de proteínas que ocurre en el parásito durante el proceso de enquistación. Aunque preliminar, este estudio es el primer reporte acerca de una enzima deubiquitinadora específica en el parásito G. intestinalis.

Este artigo apresenta uma abordagem combinada com dois objetivos. A primeira é analisar a sequência informou da enzima ubiquitina carboxil-terminal hidrolase 14 de Giardia intestinalis (UBP6) através de métodos computacionais para encontrar os componentes relacionados com a sua função hipotética. A segunda é para determinar se o gene de codificação da proteína é expressa em G. intestinalis e, se for o caso, também determinar o seu padrão de transcrição ao longo do ciclo de vida do parasita. Foi estabelecido que a proteína pertence à família de deubiquitinases Cys-dependentes e mais especificamente para proteases específicas de ubiquitina (USPs por ubiquitin specific proteases). Além disso, o centro catalítico com a tríade completo, bem como as características típicas do motivo USP também foram identificados. Uma vez que os resultados computacionais sugerem que a enzima poderia ser funcional, a transcrição reversa acoplada a PCR foi utilizado como uma primeira abordagem para determinar se, de facto, o gene codificante é expressa no parasita. Curiosamente, verificou-se não só que o gene é expresso, mas também que há uma variação de transcrição ao longo do ciclo de vida do parasita. Estes dois elementos são o ponto de partida para estudos posteriores, uma vez que tentativas sugerem que esta enzima pode estar envolvida no refill de proteínas que ocorre durante o parasita encistamento. Embora preliminares, este estudo é o primeiro relatório relativo ao estudo de uma enzima deubiquitinadora específica no parasita intestinalis.

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.