Single-cell transcriptome profiles and E−64 inhibitor data reveal the essential role of cysteine proteases in the ontogeny of Ichthyophthirius multifiliis

Article

作者: Luo, Daji ; Nie, Fanya ; Zhao, Weishan ; Zhou, Weitian ; Yang, Shiman ; Li, Ming ; Wang, Guitang ; Zou, Hong ; Li, Wenxiang

Ichthyophthiriasis (Ich), also known as white spot disease, causes significant economic losses to fish farmers once an outbreak occurs. For fish survival, it is therefore crucial to understand the pathogenic mechanism and find effective prevention methods. In this study, we obtained data for four stages (theront, trophont, protomont and tomont) of Ichthyophthirius multifiliis by single-cell RNA sequencing (scRNA-seq). We found that the invasion-related proteins encoded by highly expressed genes in the theront stage mainly belong to the leishmanolysin family proteins, heat shock proteins, transmembrane proteins and cysteine proteases (CPs). Additionally, the exosome pathway appears to play a significant role in the invasion process of the theront. Since cysteine proteases are expressed at all stages of the I. multifiliis, and five CP-related genes were significantly upregulated at the theront stage of its life cycle - two of which are enriched in the exosome pathway - we incubated I. multifiliis theronts and protomonts with cysteine protease inhibitor (E-64). Our findings revealed that E-64 could kill both stages of the parasite in vitro and affected tomont division and subsequent release. Furthermore, infection experiment showed that E-64 could significantly inhibit the invasion of theronts. Based on our preliminary analysis from the transcriptomic and E-64 experiments, we have confirmed that CPs play a crucial role in I. multifiliis. This research establishes a foundation for future strategies in the prevention and control of Ich.

2024-06-20·Nan fang yi ke da xue xue bao = Journal of Southern Medical University

[Porphyromonas gingivalis infection facilitates immune escape of esophageal cancer by enhancing YTHDF2-mediated Fas degradation].

Article

作者: Zhang, X ; Yuan, X ; Zhang, J ; Yang, Z ; Liu, Y

OBJECTIVE:

To investigate the effect of Porphyromonas gingivalis (Pg) infection on immune escape of oesophageal cancer cells and the role of YTHDF2 and Fas in this regulatory mechanism.

METHODS:

We examined YTHDF2 and Fas protein expressions in esophageal squamous cell carcinoma (ESCC) tissues with and without Pg infection using immunohistochemistry and in Pg-infected KYSE150 cells using Western blotting. The interaction between YTHDF2 and Fas was investigated by co-immunoprecipitation (Co-IP). Pg-infected KYSE150 cells with lentivirus-mediated YTHDF2 knockdown were examined for changes in expression levels of YTHDF2, cathepsin B (CTSB), Fas and FasL proteins, and the effect of E64 (a cathepsin inhibitor) on these proteins were observed. After Pg infection and E64 treatment, KYSE150 cells were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the expressions of T cell-related effector molecules were detected by flow cytometry.

RESULTS:

ESCC tissues and cells with Pg infection showed significantly increased YTHDF2 expression and lowered Fas expression. The results of Co-IP demonstrated a direct interaction between YTHDF2 and Fas. In Pg-infected KYSE150 cells with YTHDF2 knockdown, the expression of CTSB was significantly reduced while Fas and FasL expressions were significantly increased. E64 treatment of KYSE150 cells significantly decreased the expression of CTSB without affecting YTHDF2 expression and obviously increased Fas and FasL expressions. Flow cytometry showed that in Pg-infected KYSE150 cells co-cultured with PBMCs, the expressions of Granzyme B and Ki67 were significantly decreased while PD-1 expression was significantly enhanced.

CONCLUSION:

Pg infection YTHDF2-dependently regulates the expression of Fas to facilitate immune escape of esophageal cancer and thus promoting cancer progression, suggesting the key role of YTHDF2 in regulating immune escape of esophageal cancer.

2024-06-01·EUROPEAN JOURNAL OF PROTISTOLOGY

Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila

Article

作者: Arslanyolu, Muhittin ; Üstüntanır Dede, Ayça Fulya ; Dündar Orhan, Yeliz ; Duran, Şeyma

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

100 项与 E-64 相关的药物交易

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