The Molecular Basis of Ubiquitin-Conjugating Enzymes (E2s) as a Potential Target for Cancer Therapy

Ubiquitin-conjugating enzymes (E2s) are one of the three enzymes required by the ubiquitin-proteasome pathway to connect activated ubiquitin to target proteins via ubiquitin ligases. E2s determine the connection type of the ubiquitin chains, and different types of ubiquitin chains regulate the stability and activity of substrate proteins. Thus, E2s participate in the regulation of a variety of biological processes. In recent years, the importance of E2s in human health and diseases has been particularly emphasized. Studies have shown that E2s are dysregulated in variety of cancers, thus it might be a potential therapeutic target. However, the molecular basis of E2s as a therapeutic target has not been described systematically. We reviewed this issue from the perspective of the special position and role of E2s in the ubiquitin-proteasome pathway, the structure of E2s and biological processes they are involved in. In addition, the inhibitors and microRNAs targeting E2s are also summarized. This article not only provides a direction for the development of effective drugs but also lays a foundation for further study on this enzyme in the future

Molecular Characterisation and Functions of Fis1 and PDCD6 Genes from Echinococcus granulosus

Cystic echinococcosis, a parasitic zoonosis that causes significant economic losses and poses a threat to public health, is caused by larvae of the tapeworm Echinococcus granulosus. Infection causes infertile cysts in intermediate hosts that cannot produce protoscoleces (PSCs) or complete the life cycle. Herein, we cloned, expressed, and characterised mitochondrial fission protein 1 (Eg-Fis1) and programmed cell death protein 6 (Eg-PDCD6) from E. granulosus, and explored their functions related to infertile cysts. Eg-Fis1 and Eg-PDCD6 encode putative 157 and 174 residue proteins, respectively, and Western blotting indicated good reactogenicity for both. Eg-Fis1 and Eg-PDCD6 were ubiquitously distributed in all stages of E. granulosus. Furthermore, mRNAs of Eg-Fis1 and Eg-PDCD6 were upregulated following H2O2 treatment which induced apoptosis in PSCs. To investigate the regulation of apoptosis in response to oxidative stress, RNA interference (RNAi) and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays were performed. The apoptotic rate of the Eg-Fis1 RNAi group was significantly lower than non-interference group, but there was no such difference for Eg-PDCD6. In conclusion, Eg-Fis1 promotes apoptosis induced by oxidative stress, whereas Eg-PDCD6 does not appear to be a key regulator of apoptosis

An Antibody Persistent and Protective Two rSsCLP-Based Subunit Cocktail Vaccine against Sarcoptes scabiei in a Rabbit Model

Scabies is a highly contagious disease caused by Sarcoptes scabiei which burrows into stratum corneum of host’s skin. In this study, after optimizing vaccination schedule, a vaccination trial is comprised of three test groups of rabbits (n = 10/group) by immunization with (1) rSsCLP5; (2) rSsCLP12; or (3) a mixture of rSsCLP5 and rSsCLP12, three biological replicates groups (n = 10/group) and three control groups (n = 10/group). Levels of specific IgG, total IgE and cytokines in sera were detected and histopathologically analyzed as indicators of vaccine effects. The results showed that 85% (17/20) of rabbits exhibited no detectable skin lesions of S. scabiei infestation in mixed protein groups compared to single protein groups with 75% (15/20) and 70% (14/20), respectively. Moreover, the deworming rates of mixed groups are increased by 10%–20% compared with that of single groups. Each of six groups immunized with rSsCLP displayed significant increases of specific IgG, total IgE, IL-10, and TNF-α. The degree of skin damage in test groups also significantly lower than that of control groups. Thus, purified rSsCLP5 and rSsCLP12 subunit cocktail vaccine induced robust immune protection and could significantly decrease mite populations to reduce the direct transmission between rabbits

Expression Analysis and Serodiagnostic Potential of Microneme Proteins 1 and 3 in Eimeria stiedai

Eimeria stiedai is an apicomplexan protozoan parasite that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis, resulting in significant economic losses in the domestic rabbit industry. Hepatic coccidiosis lacks the typical clinical symptoms and there is a lack of effective premortem tools to timely diagnose this disease. Therefore, in the present study we cloned and expressed the two microneme proteins i.e., microneme protein 1 (EsMIC1) and microneme protein 3 (EsMIC3) from E. stiedai and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding EsMIC1 and EsMIC3 were cloned and the mRNA expression levels of these two genes at different developmental stages of E. stiedai were determined by quantitative real-time PCR analysis (qRT-PCR). The immunoreactivity of recombinant EsMIC1 (rEsMIC1) and EsMIC3 (rEsMIC3) proteins were detected by Western blotting, and indirect enzyme-linked immunosorbent assays (ELISAs) based on these two recombinant antigens were established to evaluate their serodiagnostic potential. Our results showed that the proteins encoded by the ORFs of EsMIC1 (711 bp) and EsMIC3 (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both EsMIC1 and EsMIC3 showed the highest mRNA expression levels in the merozoites stage of E. stiedai. Western blotting analysis revealed that both recombinant proteins were recognized by E. stiedai positive sera, and the indirect ELISAs using rEsMIC1 and rEsMIC3 were developed based on their good immunoreactivity, with 100% (48/48) sensitivity and 97.9% (47/48) specificity for rEsMIC1 with 100% (48/48) sensitivity and 100% (48/48) specificity for rEsMIC3, respectively. Moreover, rEsMIC1- and rEsMIC3-based indirect ELISA were able to detect corresponding antibodies in sera at days 6, 8, and 10 post E. stiedai infection, with the highest positive diagnostic rate (62.5% (30/48) for rEsMIC1 and 66.7% (32/48) for rEsMIC3) observed at day 10 post infection. Therefore, both EsMIC1 and EsMIC3 can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused by E. stiedai

Serodiagnostic Potential of Alpha-Enolase From Sarcoptes scabiei and Its Possible Role in Host-Mite Interactions

Infestation of the epidermis with the highly contagious ectoparasite, Sarcoptes scabiei, causes scabies, which is characterized by intense itching, pruritus, and secondary infection. This condition affects humans, livestock, and wildlife worldwide, incurring large economic losses and reducing the quality of human life. In the present study, we cloned the alpha-enolase, a key enzyme in the glycolytic and gluconeogenesis pathways, from S. scabiei var. cuniculi, characterized it and produced soluble recombinant enolase protein (rSsc-eno). We determined the localization of Ssc-eno in isolated mites and mites in lesioned skin. The results showed that native enolase was intensely localized in the tegument of the mouthparts, the entire legs, and the whole mites' body, as well as in the gut and reproduction system. Interestingly, we found that native enolase was widely distributed in mites in lesioned skin, with obvious high protein intensity compared with isolated mites. Building on good immunoreactivity, an indirect enzyme-linked immunosorbent assay (ELISA) based on rSsc-eno showed 92% sensitivity and 95.8% specificity, compared with other indirect ELISA in this study, rSsc-eno based ELISA is better in detecting scabies in rabbits. Besides, this method can detect S. scabiei infection as early as 1 week post infection. Compared with other detection methods, such as traditional microscopic examination and recently published universal conventional PCR, rSsc-eno ELISA was more effective to detect early infection in rabbits. Additionally, in vitro incubation experiments demonstrated the concentration-dependent acaricidal activity of rabbit anti-rSsc-eno sera against larval mites, suggested its potential as a vaccine candidate

Image_1_Serodiagnostic Potential of Alpha-Enolase From Sarcoptes scabiei and Its Possible Role in Host-Mite Interactions.TIF

<p>Infestation of the epidermis with the highly contagious ectoparasite, Sarcoptes scabiei, causes scabies, which is characterized by intense itching, pruritus, and secondary infection. This condition affects humans, livestock, and wildlife worldwide, incurring large economic losses and reducing the quality of human life. In the present study, we cloned the alpha-enolase, a key enzyme in the glycolytic and gluconeogenesis pathways, from S. scabiei var. cuniculi, characterized it and produced soluble recombinant enolase protein (rSsc-eno). We determined the localization of Ssc-eno in isolated mites and mites in lesioned skin. The results showed that native enolase was intensely localized in the tegument of the mouthparts, the entire legs, and the whole mites' body, as well as in the gut and reproduction system. Interestingly, we found that native enolase was widely distributed in mites in lesioned skin, with obvious high protein intensity compared with isolated mites. Building on good immunoreactivity, an indirect enzyme-linked immunosorbent assay (ELISA) based on rSsc-eno showed 92% sensitivity and 95.8% specificity, compared with other indirect ELISA in this study, rSsc-eno based ELISA is better in detecting scabies in rabbits. Besides, this method can detect S. scabiei infection as early as 1 week post infection. Compared with other detection methods, such as traditional microscopic examination and recently published universal conventional PCR, rSsc-eno ELISA was more effective to detect early infection in rabbits. Additionally, in vitro incubation experiments demonstrated the concentration-dependent acaricidal activity of rabbit anti-rSsc-eno sera against larval mites, suggested its potential as a vaccine candidate.</p