Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

Identification and expression analysis of a Spsb gene in planarian Dugesia japonica.

The SPSB family is comprised of four highly conserved proteins, each containing a C-terminal SOCS box motif and a central SPRY domain. Presently, Spsb genes have been found in mammals and in a few invertebrates, however, the specific functions of these genes are still unknown. In this study, we identified a Spsb gene from the planarian Dugesia japonica and termed it DjSpsb. The temporal and spatial expression patterns of DjSpsb were examined in both intact and regenerative animals, and expression levels were also quantified in response to various stressors. The results show that (1) DjSpsb is highly conserved in evolutionary history in metazoans and is at closer relationship to Spsb1, Spsb2 and Spsb4; (2) DjSpsb mRNA is mainly expressed in the head and also throughout head regeneration processes, particularly, its expression up-regulated observably on day 5 after amputation; (3) DjSpsb is also expressed in the testes and yolk glands; (4) DjSpsb expression is induced by high temperature and ethanol but inhibited by high doses of ionic liquids. The date suggests that the DjSpsb gene might be active in central nervous system (CNS) formation and functional recovery during head regeneration, and it is also involved in the development of germ cells and stress responses in the planarians.

Identification, phylogeny and expression analysis of suppressors of cytokine signaling in channel catfish.

The suppressors of cytokine signaling (SOCS) family genes play important roles in regulating a variety of signal transduction pathways that are involved in immunity, growth and development. Because of their importance, they have been extensively studied in mammalian species, but they have not been systematically studied among teleost fish species. In this study, a total of 12 SOCS genes were characterized to understand the molecular mechanisms of SOCS function in channel catfish. Phylogenetic analyses suggested that all SOCS were clustered into two main clusters. Further syntenic analysis confirmed the phylogenetic analyses and allowed the annotation of SOCS genes in channel catfish. This work, for the first time, determined the expression profiles of the 12 SOCS genes after bacterial infections with Flavobacterium columnare and Edwardsiella ictaluri in channel catfish. The SOCS1a and SOCS3a were significantly up-regulated at 4h after F. columnare challenge in the gill, but were down-regulated at later stages of pathogenesis. Similarly, SOCS1a and CISH were significantly up-regulated at 3h in intestine under E. ictaluri infection, but were down-regulated at later stages of pathogenesis at 24h and 3 days after infection. These expression patterns may indicate that SOCS genes could be induced in acute immune responses after bacterial infections, but the massive cytokine expression, especially chemokine expression after the first day of infection may have had negative feedback leading to the overall down-regulation of the expression of SOCS genes. Moreover, the differential expression patterns of SOCS genes in the catfish gill and intestine after F. columnare and E. ictaluri infection demonstrated that the regulation of SOCS gene expression was both tissue-specific and time-dependent. Taken together, these results suggested that SOCS genes were involved in immune responses to bacterial invasions, and these results set the foundation for future studies of SOCS gene functions.

Detection of endogenous SOCS1 and SOCS3 proteins by immunoprecipitation and Western blot analysis.

The suppressors of cytokine signalling (SOCS) protein family consist of eight members (SOCS 1-7, and CIS). SOCS1 and SOCS3 are the best-studied family members and have been shown to act as negative feedback inhibitors of the JAK/STAT signalling pathway. To study the physiological roles of the SOCS proteins, it is necessary to establish methods for detecting endogenous proteins often expressed at low levels in cells after cytokine induction. To facilitate the detection of endogenous SOCS1 and SOCS3 proteins, we have generated in-house antibodies specific to these proteins, which we have used together with commercially available antibodies. Here, we describe the methods for immunoprecipitating SOCS1 and SOCS3 proteins from mouse tissue extracts and their subsequent detection by Western blot analysis. These methods can also be applied to the detection of SOCS1 and SOCS3 in cell lines.

Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a.

An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26-41) alone is not enough, and that amino acids 12-41 from the N-terminus of ASB2a is essential for binding. ASB2a(12-41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12-41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated ß-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins.

The novel gene asb11: a regulator of the size of the neural progenitor compartment.

From a differential display designed to isolate genes that are down-regulated upon differentiation of the central nervous system in Danio rerio embryos, we isolated d-asb11 (ankyrin repeat and suppressor of cytokine signaling box-containing protein 11). Knockdown of the d-Asb11 protein altered the expression of neural precursor genes sox2 and sox3 and resulted in an initial relative increase in proneural cell numbers. This was reflected by neurogenin1 expansion followed by premature neuronal differentiation, as demonstrated by HuC labeling and resulting in reduced size of the definitive neuronal compartment. Forced misexpression of d-asb11 was capable of ectopically inducing sox2 while it diminished or entirely abolished neurogenesis. Overexpression of d-Asb11 in both a pluripotent and a neural-committed progenitor cell line resulted in the stimulus-induced inhibition of terminal neuronal differentiation and enhanced proliferation. We conclude that d-Asb11 is a novel regulator of the neuronal progenitor compartment size by maintaining the neural precursors in the proliferating undifferentiated state possibly through the control of SoxB1 transcription factors.