Summary
Background
Tripartite motif‐containing protein 21 (Trim21) is an E3 ubiquitin‐protein ligase that plays pivotal roles in various diseases. However, its role in mediating keratinocyte inflammation, which is a hallmark of psoriasis, has not been thoroughly elucidated.
Objectives
To clarify whether Trim21 plays a pivotal role in regulating keratinocyte inflammation in psoriasis, while focusing on identifying key Trim21 substrates involved in mediating proinflammatory cytokine and chemokine production.
Materials and methods
Cytokine and chemokine secretion was examined by quantitative real‐time polymerase chain reaction (qPCR) in Trim21‐knockdown human keratinocytes. Downstream pathways and substrates of Trim21 were evaluated using immunoblotting, immunoprecipitation and immunofluorescence. The influence of Trim21 ubiquitination on its substrates was tested by in vitro ubiquitination assay, immunoprecipitation and immunofluorescence. The effectiveness of targeting Trim21 for psoriasis treatment was assessed in vivo with haematoxylin and eosin staining, immunofluorescence and qPCR.
Results
Knocking down Trim21 expression alleviated keratinocyte inflammation. Trim21 colocalized with p65/nuclear factor (NF)‐κB in the cytosol and physically bound and ubiquitinated p65 via a lysine 63 (K63) linkage. Instead of changing p65 protein stability, Trim21 enhanced the interaction of p65 with IκB kinase, which promoted p65 phosphorylation, nuclear transport and downstream gene activation. Finally, both in vitro and in vivo experiments verified that topical application of Trim21‐specific small interfering RNA markedly ameliorated imiquimod‐induced psoriasis‐like lesions.
Conclusions
Our study confirms that upregulated Trim21 in psoriatic epidermis ubiquitylates p65 and activates the NF‐κB pathway, which promotes keratinocyte inflammation. Hence, Trim21 represents a potential target for psoriasis treatment.
Heat shock protein 16.3 (Hsp16.3) of Mycobacterium tuberculosis (MTB) containing T-cell and B-cell epitopes not only plays an important role in the survival of MTB against macrophages, but also has great potential to be used to develop new TB vaccines. In order to study whether Hsp16.3 can be replaced with its T-cell epitope for producing a vaccine against TB, we expressed and purified Hsp16.3 protein of MTB H37Rv strain and confirmed by immunoblotting. The immune responses and protection against the H37Rv induced by Hsp16.3 protein were compared with its T-cell epitope synthetic peptide in mice. The results showed that both Hsp16.3 and its synthetic peptide induced significantly stronger specific antibodies than the classical TB vaccine-BCG (bacillus Calmette-Guérin). Compared with BCG, the stimulation index in the splenolymphocyte proliferation of both recombinant protein and its synthetic peptide were remarkably enhanced, but the levels of IFN-c release were lower. Dramatic reduction in the numbers of MTB colony forming units (CFU) in the spleens and lungs was observed in the mice immunized with Hsp16.3 or its synthetic peptide. The protection provided by Hsp16.3 or its synthetic peptide in the lungs was equivalent to that provided by BCG. Both Hsp16.3 and its T-cell epitope are effective components and Hsp16.3 can be replaced with its T-cell epitope while developing the vaccine against TB, without requiring the complicated procedure of expressing and purifying Hsp16.3.
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