Elsevier

Fish & Shellfish Immunology

Volume 35, Issue 4, October 2013, Pages 1224-1234
Fish & Shellfish Immunology

Genomic structural characterization and transcriptional expression analysis of proteasome activator PA28α and PA28β subunits from Oplegnathus fasciatus

https://doi.org/10.1016/j.fsi.2013.07.040Get rights and content

Highlights

  • Genomic characterization of RbPA28α and RbPA28β showed 11 exons.

  • Putative promoter analysis showed several transcription factor binding sites.

  • In silico characterization of protein revealed conserved motifs.

  • Tissue distribution studies showed highest expression in blood.

  • mRNA profiling after LPS and poly I:C challenges showed up-regulation in immune organs.

Abstract

Proteasomes are multicatalytic subunit complexes involved in the degradation of cytosolic proteins and antigen presentation. In this study, we have characterized the alpha and beta subunits of proteasome activator complex from rock bream at the molecular level. RbPA28α and RbPA28β possessed the characteristic features of the subunits identified from mammals and teleosts. The RbPA28α and RbPA28β proteasome subunits contained a proline-rich motif (Region A), subunit-specific insert in the region corresponding to the KEKE motif of the known PA28α (Region B), conserved activation loop (Region C), a potential protein kinase C recognition site (Region D) and a highly homologous C-terminal region (Region E) among all three PA28 subunits. Multiple sequence alignment and pairwise alignment revealed that RbPA28α and RbPA28β proteins shared high homology with the teleosts and mammals. RbPA28α and RbPA28β genome possessed 11 exons interrupted by 10 introns. In silico promoter analysis of RbPA28α and RbPA28β revealed various transcription factor-binding sites displaying their regulation under various stress conditions. Tissue distribution profiling showed a higher expression in blood and gills. Transcriptional expression analysis of RbPA28α and RbPA28β showed up-regulation in the immune tissues following LPS and poly I:C challenges, providing further evidence for the immunological role of RbPA28α and RbPA28β.

Introduction

Proteasomes are large multi-subunit self-compartmentalizing proteolytic complexes comprising a central catalytic machine (20S proteasome) capped by two terminal regulatory sub-complexes known as 19S (PA700) or PA28. The proteasomes are made of four stacked heptameric rings formed by genetically and structurally similar two α-type and two β-type subunits in a α7β7β7α7 pattern. The subunits are axially stacked on top of each other forming a cylindrical barrel-like structure. In order to avoid unwanted damage of the cytosolic proteins, the 20S channel is usually closed by a gate formed from the N-termini of the α-subunits. The regulatory particle plays a significant role in identifying the ubiquitinated proteins and potential substrates directed for proteolysis. Upon binding of the regulatory complex (19S or PA28), the gate opens allowing the creation/formation of a conglomerate of PA28-20S-PA28 (or 19S-20S-19S) thus activating the latent central catalytic core. The catalytic sites are localized in some of the β-subunits belonging to the central core. Upon interferon-γ (IFN-γ) signaling, three of the seven constitutively expressed β-subunits in the inner ring are replaced by three inducible β-subunits forming an immunoproteasome which is responsible for the generation of peptides that can bind the major histocompatibility complex (MHC) class I molecules [1], [2].

The eukaryotic 11S regulators (11S REG or PA26 or PA28) comprise three isoforms called PA28α, PA28β, and PA28γ (also called REGα, REGβ, and REGγ or PSME1, PSME2 and PSME3, respectively). One of the 11S REG forms a heteroheptameric ring composed of IFN-γ-inducible α and β subunits, while the other ring is composed of homo-hexameric PA28γ. The 11S REG activates the degradation of peptides in an ATP and ubiquitination-independent fashion unlike the 19S regulators that are ATP-dependent and degrade ubiquitinylated proteins. PA28α and PA28β are immunologically significant as they are involved in the activation of proteasomes and degradation of cellular proteins resulting in peptides which bind to the major histocompatibility complex MHC class I molecules facilitating antigen presentation [3], [4], [5], [6], [7].

Studies on antigen presentation mechanisms and activation of adaptive immunity have gained significance in fish and the molecules involved in these processes are being extensively investigated. Although PA28α, β, and γ subunits have been largely explored in human, mice and model organisms like zebrafish, few reports are available that have characterized the PA28α/β subunits and further substantiated their involvement in antigen presentation in fish [8], [9], [10]. Moreover, only a few reports are available on the genomic characterization of the proteasome activators. Rock bream are economically significant commercial species in Korea and are prone to infections by various pathogens like iridovirus, and Edwardsiella tarda [11], [12], [13] causing mass mortality and heavy economic loss. In order to employ novel strategies like vaccination for the prevention of such deadly diseases, it is essential to understand their antigen presentation mechanisms and adaptive immune system. In the present study, we have characterized the PA28α and PA28β subunits from rock bream at the genomic level.

Section snippets

Rock bream cDNA and BAC library construction, identification of PA28α and PA28β genes

The rock bream cDNA and BAC library were custom constructed as described in our earlier reports [14]. A search for genes related to immunity in the cDNA database using BLAST led to identification of two full-length cDNA contigs that shared a high homology with PA28α and PA28β homologs available in the NCBI database. They were designated as RbPA28α and RbPA28β and considered for further studies. Two BAC clones harboring RbPA28α and RbPA28β were identified from the BAC library using the gene

In-silico characterization of RbPA28α and RbPA28β

A comparison of the characteristic features of RbPA28α and RbPA28β (Table 1) showed that RbPA28α possesses an ORF of 753 bp coding for 250 amino acids and RbPA28β possesses an ORF of 738 bp coding for 245 amino acids. Both RbPA28α and RbPA28β cDNA possessed a single polyadenylation signal (Table 1) unlike the yellow croaker and flounder PA28β [9], [10]. Interestingly, both RbPA28α and RbPA28β contained a proline rich motif belonging to region A (Table 1) and the monomers were composed of four

Conclusion

In this study, we have characterized the α and β subunits of rock bream proteasome activator complex at the genome level and further provided evidence for the transcriptional modulations triggered by an immune stimulus. The evidences suggest that these subunits may be involved in the adaptive immunity of rock bream.

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