169,830 research outputs found
Structural analysis of SHARPIN, a subunit of a large multi-protein E3 ubiquitin ligase, reveals a novel dimerization function for the pleckstrin homology superfold
No full textSHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold
Current model of SHARPIN function.
No full text<p>(A) SHARPIN inhibits integrin activation by binding to the α-integrin cytoplasmic domain and preventing binding of the integrin activator TALIN. In addition, SHARPIN is part of LUBAC, which is required for activation of the canonical NF-κB pathway. We now demonstrate that integrin and RNF31 binding are mutually exclusive as they are mediated by partially overlapping binding sites within the SHARPIN UBL domain. (B) An overview of SHARPIN interactions and how these interactions affect diverse signaling pathways. SHARPIN is depicted schematically with its functional domains (an N-terminal PH domain, a central UBL domain and a C-terminal NZF domain). The UBL domain is a multi-faceted protein interaction hub that has been shown to interact with a number of proteins, highlighted in yellow bars. These interactions each have specific functional consequences, highlighted in green bars. In addition, the N-terminal PH domain of SHARPIN mediates SHARPIN homodimerization and the C-terminal NZF domain is required for LUBAC function.</p
Designing SHARPIN mutants using a SHARPIN UBL domain model.
No full text<p>(A) Superposition of the SHARPIN UBL domain model backbone (purple) with the UBL domain of HOIL1L (Green, PDB accession code: 4DBG). (B) Surface representation of the SHARPIN UBL model. The surface is color-coded according to residue hydrophobicity from light to deep purple. Footprints of residues mutated in this study are indicated as green outlines. (C) Alignment of part of the SHARPIN UBL domain across different species. Conserved residues mutated in this study are indicated in red.</p
Fine mapping of the RNF31 binding site in SHARPIN.
No full text<p>(A) Western blot analysis of SHARPIN and β-tubulin levels in control- or SHARPIN-silenced PC3 cells. (B) TNF-induced NF-κB promoter activity of SHARPIN- or control-silenced PC3 cells was measured using a luciferase reporter assay. (n = 3 with 5 replicates each). (C) TNF-induced NF-κB promoter activity of SHARPIN-silenced PC3 cells, expressing GFP alone, WT or mutant GFP-SHARPIN (n = 6–15 measurements from 2–3 experiments). (D,E) Interaction between RNF31 and WT or mutant GST-SHARPIN was determined using an ELISA-based binding assay (n = 3) (D) or Far-Western analysis (E). All numerical data are mean ± s.e.m. ***: p<0.001, **: p<0.01, *: p<0.05.</p
<i>Sharpin</i> expression in mouse embryonic fibroblasts.
No full text<p>Transfection of <i>Flag</i> tagged <i>Sharpin</i> construct or shRNA lentivirus infection were respectively applied to mouse fibroblasts. Green fluorescent protein (GFP) labeled lentivirus were used as a control to monitor the infect efficacy. (<b>A</b>) Fluorescence detection of MEFs with co-transfection of both <i>Sharpin</i> shRNA lentivirus and GFP labeled lentivirus; (<b>B</b>) Differential interference micrograph; (<b>C</b>) Merged image of A and B; (<b>D</b>) Real-time PCR quantification of <i>Sharpin</i> in MEFs infected by <i>Sharpin</i>-shRNA or transfected by <i>Sharpin</i>-flag construct; (<b>E</b>) Anti-FLAG Western blot after anti-FLAG affinity gel immunoprecipitation of extracts from <i>Sharpin</i>-flag transfected MEFs. (<b>F</b>) Sequence data for the <i>Flag</i> tagged <i>Sharpin</i> construct.</p
Sharpin interacts with ubiquitin and the E3 ubiquitin ligase Hoip.
No full text<p>A. Domain structure of HOIL-1L, Hoip and Sharpin. Sharpin is similar to HOIL-1L in its Ubl and NZF domains but lacks the Ring/IBR/Ring E3 ligase motif. For Hoip and Sharpin, positions used to generate deletion constructs are indicated. B. The NZF domain of Sharpin binds ubiquitin. An immobilized GST fusion protein of the NZF domain (or GST alone) was incubated with linear, K48 or K63 linked polyubiquitin chains; after centrifugation and washing, input and precipitate samples were analyzed by Western blotting with anti-ubiquitin. C. The Ubl domain of Sharpin binds to Hoip. GFP-fusion proteins of Sharpin or fragments thereof were coexpressed with Flag-tagged Hoip in HEK293 cells. Cell lysates were subjected to immunoprecipitation of GFP containing proteins using GFP-Trap matrix, and input (left) and precipitate (right) samples were analyzed by Western blotting using anti-GFP (upper panels) and anti-Flag (lower panels) antibodies. Note that the isolated Ubl domain (residues 171–310) of Sharpin precipitates Hoip very efficiently despite low expression levels. D. The central region of Hoip mediates interaction with Sharpin. Flag-tagged fragments of Hoip were coexpressed with GFP-Sharpin, and subjected to immunoprecipitation of GFP, as in C. E. The UBA domain of Hoip is sufficient for interaction with Sharpin. Myc-tagged Sharpin was coexpressed with a GFP-Hoip encompassing the UBA domain, or GFP alone. Cell lysates were subjected to immunoprecipitation of GFP and input (in) and precipitate (p) samples were analyzed by Western blotting. F. Interaction in hepatocarcinoma cells. Constructs encoding GFP as a fusion with full length Sharpin (amino acids 1–382), or only the N-terminal region (residues 1–171) were expressed in Huh-7 hepatocarcinoma cells. After cell lysis, GFP-containing proteins were immunoprecipitated using GFP-Trap matrix. Input (in) and precipitate (p) samples were analyzed by Western blotting using antibodies against human Hoip (upper panel) or GFP (lower panel).</p
68Ga-DOTA-E[c(RGDfK)]2 PET Imaging of SHARPIN-Regulated Integrin Activity in Mice
No full textShank-associated RH domain-interacting protein (SHARPIN) is a cytosolic protein that plays a key role in activation of nuclear factor k-light-chain enhancer of activated B cells and regulation of inflammation. Furthermore, SHARPIN controls integrin-dependent cell adhesion and migration in several normal and malignant cell types, and loss of SHARPIN correlates with increased integrin activity in mice. Arginyl-glycyl-aspartic acid (RGD), a cell adhesion tripeptide motif, is an integrin recognition sequence that facilitates PET imaging of integrin upregulation during tumor angiogenesis. We hypothesized that increased integrin activity due to loss of SHARPIN protein would affect the uptake of alpha(v beta)3-selective cyclic, dimeric peptide Ga-68-DOT-AE[c(RGDfK)] 2, where E[c(RGDfk)] 2 5 glutamic acid-[cyclo(arginyl-glycylaspartic acid-D-phenylalanine-lysine)], both in several tissue types and in the tumor microenvironment. To test this hypothesis, we used RGD-based in vivo PET imaging to evaluate wild-type (wt) and SHARPIN-deficient mice (Sharpincpdm, where cpdm 5 chronic proliferative dermatitis in mice) with and without melanoma tumor allografts. Methods: Sharpincpdm mice with spontaneous null mutation in the Sharpin gene and their wt littermates with or without B16-F10-luc melanoma tumors were studied by in vivo imaging and ex vivo measurements with cyclic-RGD peptide Ga-68-DOTA-E[c(RGDfK)](2). After the last Ga-68-DOTA-E[c(RGDfK)](2) peptide PET/CT, tumors were cut into cryosections for autoradiography, histology, and immunohistochemistry. Results: The ex vivo uptake of Ga-68-DOTAE[ c(RGDfK)](2) in the mouse skin and tumor was significantly higher in Sharpincpdm mice than in wt mice. B16-F10-luc tumors were detected 4 d after inoculation, without differences in volume or blood flow between the mouse strains. PET imaging with Ga-68-DOTAE[ c(RGDfK)](2) peptide at day 10 after inoculation revealed significantly higher uptake in the tumors transplanted into Sharpincpdm mice than in wt mice. Furthermore, tumor vascularization was increased in the Sharpincpdm mice. Conclusion: Sharpincpdm mice demonstrated increased integrin activity and vascularization in B16-F10-luc melanoma tumors, as demonstrated by RGD-based in vivo PET imaging. These data indicate that SHARPIN, a protein previously associated with increased cancer growth and metastasis, may also have important regulatory roles in controlling the tumor microenvironment.</p
The UBL domain of SHARPIN mediates binding to integrin.
No full text<p>(A) Schematic representation of SHARPIN with its functional domains and the SHARPIN fragments used in this study. (B) Pull-down experiments to determine the interaction between GFP-SHARPIN (full-length or fragments) and peptides corresponding to the cytoplasmic domain of ITGAL and ITGB2. (C) Far-Western analysis of GST-SHARPIN (full-length or fragments) binding to full-length ITGAL-ITGB2 or ITGAL-ITGB2 lacking both cytoplasmic tails. Loading controls for GST-SHARPIN (full-length or fragments) and both ITGAL-ITGB2s are shown. (D) Fluorescence polarization-based titration of GST-SHARPIN (full-length or fragments) binding to an integrin peptide corresponding to the conserved domain within the cytoplasmic tail of ITGA2. Average normalized binding curves are shown (mean ± s.e.m. ***: p<0.001).</p
SHARPIN negatively associates with TRAF2-mediated NFκB activation.
No full textNFκB is an inducible transcriptional factor controlled by two principal signaling cascades and plays pivotal roles in diverse physiological processes including inflammation, apoptosis, oncogenesis, immunity, and development. Activation of NFκB signaling was detected in skin of SHAPRIN-deficient mice and can be diminished by an NFκB inhibitor. However, in vitro studies demonstrated that SHARPIN activates NFκB signaling by forming a linear ubiquitin chain assembly complex with RNF31 (HOIP) and RBCK1 (HOIL1). The inconsistency between in vivo and in vitro findings about SHARPIN's function on NFκB activation could be partially due to SHARPIN's potential interactions with downstream molecules of NFκB pathway. In this study, 17 anti-flag immunoprecipitated proteins, including TRAF2, were identified by mass spectrum analysis among Sharpin-Flag transfected mouse fibroblasts, B lymphocytes, and BALB/c LN stroma 12 cells suggesting their interaction with SHARPIN. Interaction between SHARPIN and TRAF2 confirmed previous yeast two hybridization reports that SHARPIN was one TRAF2's partners. Furthermore, luciferase-based NFκB reporter assays demonstrated that SHARPIN negatively associates with NFκB activation, which can be partly compensated by over-expression of TRAF2. These data suggested that other than activating NFκB signaling by forming ubiquitin ligase complex with RNF31 and RBCK1, SHARPIN may also negatively associate with NFκB activation via interactions with other NFκB members, such as TRAF2
SHARPIN is an endogenous inhibitor of beta1-integrin activation.
No full textRegulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate beta1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of beta1-integrins in an RNAi screen. SHARPIN inhibited beta1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased beta1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin alpha-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of beta1-integrins from inactive to active conformations
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