Characterization of Sharpin 
Orthologs of SHARPIN protein are found in various species, including human, mouse and rat. Motif prediction programming, using COILS [11] and MotifScan [12], suggests that SHARPIN contains a coiled-coil (CC) domain, a ubiquitin-like (UBL) domain, and a zinc-finger Ran-binding protein 2 (ZFRBP) domain. These functional motifs constitute similar domain profiles that are present in the SHARPIN protein of all three origins (Fig. 1A), suggesting that SHARPIN exerts highly conserved functions across species. Spontaneous mutations in the mouse Sharpin gene results in a complex inflammatory phenotype characterized by severe dermatitis (Fig. 1B), systemic inflammation and an enlarged spleen (Fig. 1C) caused by extramedullary hematopoiesis [3]. The endogenous expression of Sharpin mRNA in BMDC was determined by quantitative real time-PCR (qRT-PCR) following culture in medium only or after stimulation with LPS. Sharpin mRNA was present in BMDC generated from WT mice (Fig. 1D) and its level was modestly decreased by LPS stimulation. There was a significant reduction of Sharpin mRNA (6–7-fold) in BMDC generated from cpdm mice. Transfection of Flag-tagged Sharpin in fibroblasts (NIH3T3) and macrophages (RAW264.7) indicated cytoplasmic localization of the SHARPIN protein (Fig. 1E).
10.1371/journal.pone.0031809.g001 Figure 1  In vivo and in vitro features of SHARPIN.
(A) COILS and MotifScan programs were used to predict the presence of CC (coiled-coil) domain, UBL (ubiquitin-like) domain and ZFRBP (zinc-finger Ran-Binding protein 2) domain which form similar motif patterns in the SHARPIN protein of human, mouse and rat origins. (B) Eight week-old females of WT and cpdm mice. The mutant mice (above) develop progressive skin inflammation starting at about four weeks. (C) Extramedullary hematopoiesis causes marked enlargement of the spleen of cpdm mice. (D) BMDC were incubated in the absence or presence of 100 ng/ml LPS for 4 hours. The mRNA level of Sharpin was measured by qRT-PCR and presented relative to the mRNA expression in non-stimulated WT BMDC. Bars represent the mean ± s.d. of 3 mice. * P<0.05; ** P<0.001. (E) Fibroblast (NIH3T3) and macrophages (RAW264.7) cells were transfected with the expression plasmid pFLAG-SHARPIN. After 48 hours, cells were fixed and probed with anti-FLAG and FITC-conjugated secondary antibody. Nuclei were stained with DAPI. In both transfected cell lines, FLAG-SHARPIN was found to be cytoplasm-localized.

P