RESUMO
Study of CD4+ T cell response and T cell receptor (TCR) specificity is crucial for understanding etiology of immune-mediated diseases and developing targeted therapies. However, solubility, accessibility, and stability of synthetic antigenic peptides used in T cell assays may be a critical point in such studies. Here we present a T cell activation reporter system using recombinant proteins containing antigenic epitopes fused with bacterial thioredoxin (trx-peptides) and obtained by bacterial expression. We report that co-incubation of CD4+ HA1.7 TCR+ reporter Jurkat 76 TRP cells with CD80+ HLA-DRB1*01:01+ HeLa cells or CD4+ Ob.1A12 TCR+ Jurkat 76 TRP with CD80+ HLA-DRB1*15:01+ HeLa cells resulted in activation of reporter Jurkat 76 TPR after addition of recombinant trx-peptide fusion proteins, containing TCR-specific epitopes. Trx-peptides were comparable with corresponding synthetic peptides in their capacity to activate Jurkat 76 TPR. These data demonstrate that thioredoxin as a carrier protein (trx) for antigenic peptides exhibits minimal interference with recognition of MHC-specific peptides by TCRs and consequent T cell activation. Our findings highlight potential feasibility of trx-peptides as a reagent for assessing the immunogenicity of antigenic fragments.
RESUMO
Ribosome biogenesis is consecutive coordinated maturation of ribosomal precursors in the nucleolus, nucleoplasm, and cytoplasm. The formation of mature ribosomal subunits involves hundreds of ribosomal biogenesis factors that ensure ribosomal RNA processing, tertiary structure, and interaction with ribosomal proteins. Although the main features and stages of ribosome biogenesis are conservative among different groups of eukaryotes, this process in human cells has become more complicated due to the larger size of the ribosomes and pre-ribosomes and intricate regulatory pathways affecting their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. A previous part of this review summarized recent data on the processing of the primary rRNA transcript and compared the maturation of the small 40S subunit in yeast and human cells. This part of the review focuses on the biogenesis of the large 60S subunit of eukaryotic ribosomes.
RESUMO
The formation of eukaryotic ribosomes is a sequential process of ribosomal precursors maturation in the nucleolus, nucleoplasm, and cytoplasm. Hundreds of ribosomal biogenesis factors ensure the accurate processing and formation of the ribosomal RNAs' tertiary structure, and they interact with ribosomal proteins. Most of what we know about the ribosome assembly has been derived from yeast cell studies, and the mechanisms of ribosome biogenesis in eukaryotes are considered quite conservative. Although the main stages of ribosome biogenesis are similar across different groups of eukaryotes, this process in humans is much more complicated owing to the larger size of the ribosomes and pre-ribosomes and the emergence of regulatory pathways that affect their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. This review addresses the key aspects of yeast and human ribosome biogenesis, using the 40S subunit as an example. The mechanisms underlying these differences are still not well understood, because, unlike yeast, there are no effective methods for characterizing pre-ribosomal complexes in humans. Understanding the mechanisms of human ribosome assembly would have an incidence on a growing number of genetic diseases (ribosomopathies) caused by mutations in the genes encoding ribosomal proteins and ribosome biogenesis factors. In addition, there is evidence that ribosome assembly is regulated by oncogenic signaling pathways, and that defects in the ribosome biogenesis are linked to the activation of tumor suppressors.
