SHANK-associated RH Domain-interacting Protein Research Articles

Attenuation of HOIL-1L ligase activity promotes systemic autoimmune disorders by augmenting linear ubiquitin signaling.

Linear ubiquitin chains, which are generated specifically by the linear ubiquitin assembly complex (LUBAC) ubiquitin ligase, play crucial roles in immune signaling, including NF-κB activation. LUBAC comprises catalytic large isoform of heme-oxidized iron regulatory protein 2 ubiquitin ligase 1 (HOIL-1L) interacting protein (HOIP), accessory HOIL-1L, and SHANK-associated RH domain-interacting protein (SHARPIN). Deletion of the ubiquitin ligase activity of HOIL-1L, an accessory ligase of LUBAC, augments LUBAC functions by enhancing LUBAC-mediated linear ubiquitination, which is catalyzed by HOIP. Here, we show that HOIL-1L ΔRING1 mice, which exhibit augmented LUBAC functions upon loss of the HOIL-1L ligase, developed systemic lupus erythematosus (SLE) and Sjögren's syndrome in a female-dominant fashion. Augmented LUBAC activity led to hyperactivation of both lymphoid and myeloid cells. In line with the findings in mice, we sought to identify missense single nucleotide polymorphisms/variations of the RBCK1/HOIL-1L gene in humans that attenuate HOIL-1L ligase activity. We found that the R464H variant, which is encoded by rs774507518 within the RBCK1/HOIL-1L gene, attenuated HOIL-1L ligase activity and augmented LUBAC-mediated immune signaling, including that mediated by Toll-like receptors. We also found that rs774507518 was enriched significantly in patients with SLE, strongly suggesting that RBCK1/HOIL-1L is an SLE susceptibility gene and that augmented linear ubiquitin signaling generated specifically by LUBAC underlies the pathogenesis of this prototype systemic autoimmune disease.

Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity

Attachment of polyubiquitin (poly-Ub) chains to proteins is a major posttranslational modification in eukaryotes. Linear ubiquitin chain assembly complex, consisting of HOIP (HOIL-1-interacting protein), HOIL-1L (heme-oxidized IRP2 Ub ligase 1), and SHARPIN (Shank-associated RH domain-interacting protein), specifically synthesizes "head-to-tail" poly-Ub chains, which are linkedviathe N-terminal methionine α-amino and C-terminal carboxylate of adjacent Ub units and are thus commonly called "linear" poly-Ub chains. Linear ubiquitin chain assembly complex-assembled linear poly-Ub chains play key roles in immune signaling and suppression of cell death and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear poly-Ubviaits Npl4 zinc finger (NZF) domain. Although the structure of the bound form of the HOIL-1L NZF domain with linear di-Ub is known, several aspects of the recognition specificity remain unexplained. Here, we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear di-Ub-bound state while rejecting other potential Ub species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear Ub binding, and interactions between the NZF core and tail revealed conserved electrostatic contacts, which were sensitive to charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear Ub affinity by weakening the integrity of the linear di-Ub-bound conformation. The described molecular determinants of linear di-Ub binding provide insight into the dynamic aspects of the Ub code and the NZF domain's role in full-length HOIL-1L.

WITHDRAWN: Overexpression of SHARPIN promotes tumor progression in ovarian cancer

SHARPIN (Shank-associated RH domain interacting protein) plays an important role in tumorigenesis. However, its role in ovarian cancer remains largely unknown. To investigate this issue, we systematically analyzed the amplification and expression of the SHARPIN in the TCGA database. From the database, we found that SHARPIN was amplified in ovarian cancer compared to normal ovarian tissue, and the mRNA level of SHARPIN was significantly elevated in ovarian cancer compared to non-tumorigenic ovarian tissue. In addition, we observed similar results from ovarian cancer cell lines and clinical samples from ovarian cancer patients, which indicated that increased SHARPIN expression is associated with tumorigenesis in ovarian cancer. SHARPIN knockdown inhibited the migration and invasion of ovarian cancer cells, also inhibited cell cycle and promoted apoptosis, thereby suppressing cell proliferation. RNA-seq results showed that SHARPIN significantly increased the expression of P53 and P21 and decreased the expression of Cyclin D1 and c-Myc, all of which are involved in the regulation of cell proliferation. Subsequent mechanistic exploration revealed that SHARPIN knockdown increased the expression of caspases 3 and 9, leading to apoptosis of ovarian cancer cells. We also found that high expression of SHARPIN was associated with poor prognosis of ovarian cancer patients. Collectively, we demonstrated a positive correlation between SHARPIN and ovarian cancer progression and provide a basis for combined targeted therapy strategies for future ovarian cancer treatment.

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbβ3 and LUBAC

AbstractPlatelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

SHARPIN regulates the development of clear cell renal cell carcinoma by promoting von Hippel-Lindau protein ubiquitination and degradation.

SHANK‐associated RH domain interacting protein (SHARPIN) plays an important role in carcinogenesis, as well as inflammation and immunity. Our study explored the effects and underlying mechanisms of SHARPIN in clear cell renal cell carcinoma (ccRCC). By analyzing The Cancer Genome Atlas database, we found that upregulated SHARPIN in patients with ccRCC led to a poor prognosis. Semiquantitative immunohistochemical analysis of clinical samples was carried out and the results suggested the positive association between SHARPIN and hypoxia‐induced factor‐2α (HIF‐2α). Von Hippel‐Lindau protein (pVHL) is a tumor suppressor that contributes to degrading HIF‐2α. Mechanically, SHARPIN promoted the ubiquitination and proteasomal degradation of pVHL, resulting in the sustained activation of HIF‐2α. The α and β domains of pVHL and ubiquitin‐like domain of SHARPIN are required for the interaction. The knockdown of SHARPIN effectively inhibited acquired sorafenib resistance in ccRCC cell lines and tumor growth in xenograft models. In conclusion, our work reveals a novel posttranslational regulation of SHARPIN on pVHL, indicating that SHARPIN could be a potential target for ccRCC treatment.

Downregulation of SHANK-associated RH domain-interacting protein elevates interleukin-33 expression by stimulating the Janus kinase 2/signal transducer and activator of transcription signalling pathway in HaCaT cells.

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which T-helper type 2 (Th2) immune responses are dominant. SH3 and multiple ankyrin repeat domains (SHANK)-associated RH domain-interacting protein (SHARPIN)is expressed at low levels in AD, resulting in the upregulation of the signal transducer and activator of transcription (STAT)3 protein and the Th2 cytokine, interleukin (IL)-33. However, the roles of SHARPIN in AD are not yet fully elucidated. To evaluate the signalling interactions of SHARPIN and IL-33 in order to improve understanding of AD pathogenesis. Western blotting was used to detect the Janus kinase (JAK)/STAT signalling proteins and IL-33 protein in HaCaT cells to determine the key proteins mediating the interaction between SHARPIN and IL-33. The findings were validated by immunofluorescence and immunohistochemical staining. Chromatin immunoprecipitation assays were used to evaluate the activity of STAT3 at theIL-33promoter. We found that phosphorylated (p)JAK2 and pSTAT3 were upregulated in SHARPIN-knockdown HaCaT cells. Subsequent chromatin immunoprecipitation assays revealed that STAT3 binds to the IL-33 promoter to mediate IL-33 expression. Moreover, SHARPIN-mediated expression of IL-33 was reduced after treatment of HaCaT cells with the JAK/STAT inhibitor ruxolitinib. STAT3 and IL-33 expression levels were higher in AD skin lesion tissues than in normal skin tissues. These findings suggest that SHARPIN modulates inflammation in HaCaT cellsby inhibiting JAK/STAT signalling, supporting the application of SHARPIN as a potential therapeutic target for AD.

SHARPIN: Role in Finding NEMO and in Amyloid-Beta Clearance and Degradation (ABCD) Pathway in Alzheimer's Disease?

SHANK- associated RH domain-interacting protein (SHARPIN) is a multifunctional protein associated with numerous physiological functions and many diseases. The primary role of the protein as a LUBAC-dependent component in regulating the activation of the transcription factor NF-κB accounts to its role in inflammation and antiapoptosis. Hence, an alteration of SHARPIN expression or genetic mutations or polymorphisms leads to the alteration of the above-mentioned primary physiological functions contributing to inflammation-associated diseases and cancer, respectively. However, there are complications of targeting SHARPIN as a therapeutic approach, which arises from the wide-range of LUBAC-independent functions and yet unknown roles of SHARPIN including neuronal functions. The identification of SHARPIN as a postsynaptic protein and the emerging studies indicating its role in several neurodegenerative diseases including Alzheimer's disease suggests a strong role of SHARPIN in neuronal functioning. This review summarizes the functional roles of SHARPIN in normal physiology and disease pathogenesis and strongly suggests a need for concentrating more studies on identifying the unknown neuronal functions of SHARPIN and hence its role in neurodegenerative diseases.

A novel role for SHARPIN in amyloid-β phagocytosis and inflammation by peripheral blood-derived macrophages in Alzheimer's disease

Defective immune cell-mediated clearance of amyloid-beta (Aβ) and Aβ-associated inflammatory activation of immune cells are key contributors in pathogenesis of Alzheimer's disease (AD). However, the underlying mechanisms remain elusive. Shank-associated RH domain-interacting protein (SHARPIN) is a critical regulator of inflammatory response. Using in vitro cultures of THP-1-derived macrophages exposed to Aβ and AD patient–derived macrophages, we demonstrate the role of SHARPIN as an obligate regulator of Aβ phagocytosis and inflammation in macrophages. Specifically, Aβ-stimulated SHARPIN in THP-1 macrophages promoted Aβ phagocytosis and expression of proinflammatory markers. In addition, Aβ-stimulated SHARPIN in macrophages promoted neuronal cell-death in differentiated SHSY5Y neurons. Furthermore, we report a novel regulatory link between SHARPIN and the NLRP3 inflammasome in response to Aβ in THP-1 macrophages. In line with our in vitro observations, a strong positive association was demonstrated between levels of Aβ42 in blood plasma of mild cognitive impairment and AD patients with SHARPIN expression in macrophages obtained from respective patient-derived peripheral blood mononuclear cells. Together, our findings show SHARPIN as a critical determinant in mediating macrophage response to Aβ and pathogenesis of AD.

SHARPIN regulates cell proliferation of cutaneous basal cell carcinoma via inactivation of the transcriptional factors GLI2 and c‑JUN.

SHANK-associated RH domain-interacting protein (SHARPIN) is a component of the linear ubiquitin chain assembly complex that can enhance the NF-κB and JNK signaling pathways, acting as a tumor-associated protein in a variety of cancer types. The present study investigated the role of SHARPIN in cutaneous basal cell carcinoma (BCC). Human BCC (n=26) and normal skin (n=5) tissues, and BCC (TE354.T) and normal skin (HaCaT) cell lines were used to evaluate SHARPIN expression level using immunohistochemistry and western blotting, respectively. A lentivirus carrying SHARPIN-targeting or negative control short hairpin RNA was infected into TE354.T cells, and the infected stable cells were assayed to analyze tumor cell proliferation, cell cycle, apoptosis, migration and invasion by Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine incorporation assays, flow cytometry and Transwell assays. Western blotting was performed to assess the protein expression levels of gene signaling in SHARPIN-silenced BCC cells. SHARPIN protein expression levels were downregulated or absent in BCC cancer nests and precancerous lesions compared with normal skin samples. In addition, SHARPIN expression levels were lower in TE354.T cells compared with HaCaT cells. SHARPIN shRNA enhanced tumor cell proliferation and the S phase of the cell cycle, whereas BCC cell apoptotic rates, and migratory and invasive abilities were not significantly altered. The expression levels of cyclin D1, cyclin-dependent kinase 4, phosphorylated-c-JUN and GLI family zinc finger 2 proteins were increased, whereas Patched 1 (PTCH1) and PTCH2 were decreased in the SHARPIN-shRNA-infected BCC cells. Therefore, the present results suggested that SHARPIN may act as a tumor suppressor during BCC development.

SHARPIN overexpression promotes TAK1 expression and activates JNKs and NF-κB pathway in Mycosis Fungoides.

Mycosis Fungoides (MF) is the most common subtype of cutaneous T-cell lymphomas (CTCL). Shank-associated RH domain-interacting protein (SHARPIN) participates in the initiation and development of multiple tumors. However, the clinical significance of SHARPIN in MF hasn't been investigated. The c-Jun N-terminal kinases (JNKs) pathway is a member of mitogen-activated protein kinases (MAPKs). Its dysregulation is observed in various tumors including CTCL, whereas the roles of JNKs pathway in MF remain largely unknown, the relationship between SHARPIN and JNKs pathway remains elusive. Herein, we showed that upregulated expression of SHARPIN was related to poor prognosis of MF patients. In vitro experiments found increased SHARPIN expression and activation of JNKs pathway in MF cell line MyLa2059. SHARPIN induced transforming growth factor β activated kinase-1 (TAK1) transcription, which is an upstream kinase of JNKs, NF-κB and p38 pathway, leading to activation of JNKs and NF-κB pathway. SHARPIN also promoted p38 signalling independent of TAK1 expression, by which overexpression of SHARPIN induced cell proliferation, inhibited apoptosis, enhanced migration and invasion of MyLa2059. Our work provided direct evidences for effects of SHARPIN on JNKs and NF-κB pathway, and the contributing roles of JNKs, NF-κB and p38 pathway regulated by SHARPIN in the development of MF.

SHARPIN Promotes Melanoma Progression via Rap1 Signaling Pathway

SHARPIN, as a tumor-associated gene, is involved in the metastatic process of many kinds of tumors. Herein, we studied the function of Shank-associated RH domain interacting protein (SHARPIN) in melanoma metastasis and the relevant molecular mechanisms. We found that SHARPIN expression was increased in melanoma tissues and activated the process of proliferation, migration, and invasion invitro and invivo, resulting in a poor prognosis of the disease. Functional analysis demonstrated that SHARPIN promoted melanoma migration and invasion by regulating Ras-associated protein-1(Rap1) and its downstream pathways, including p38 and JNK/c-Jun. Rap1 activator (8-pCPT-2'-O-Me-cAMP) and inhibitor (ESI-09 and farnesylthiosalicylic acid-amide) treatments could partially rescue invasion and migration of tumor cells. Additionally, SHARPIN expression in cell lines and public datasets also indicated that molecules other than BRAF and N-RAS may contribute to SHARPIN activation. In conclusion, our broad-in-depth work suggests that SHARPIN promotes melanoma development via p38 and JNK/c-Jun pathways through upregulation of Rap1 expression.

MyD88 mediates the pathogenesis of dermatitis in SHARPIN-deficient mice by regulating TNF production on the skin

Abstract Mice deficient in SHANK-associated RH domain–interacting protein (SHARPIN), a component of the linear ubiquitin chain assembly complex (LUBAC), develop a spontaneous inflammatory disorder with pathologic hallmarks similar to atopic dermatitis and psoriasis in humans. Previous studies identified the crucial role of components of the TNF and IL-1 signaling pathways in the progression of disease in SHARPIN-deficient mice. However, an innate adaptor or sensor that relates to the disease progression has remained unknown. In this study, we found that the genetic ablation of myeloid differentiation primary response 88 (MyD88) completely rescued skin inflammation in SHARPIN-deficient (Sharpincpdm) mice. Systemic inflammation and immune cell dysregulation were also rescued. Fibroblasts derived from SharpincpdmMyd88−⁄− micefailed to provide protection against TNF-induced cell death. SharpincpdmMyd88−⁄− mice had reduced TNF production in their skin. Furthermore, depletion of the microbiota through the oral administration of antibiotics partially rescued both the skin inflammation and systemic inflammation, demonstrating a role for the gut microbiota in SHARPIN-deficient mice. Our findings suggest a detrimental role for the innate immune adaptor MyD88 in instigating skin inflammation in Sharpincpdm mice.

68Ga-DOTA-E[c(RGDfK)]2 PET Imaging of SHARPIN-Regulated Integrin Activity in Mice.

Shank-associated RH domain-interacting protein (SHARPIN) is a cytosolic protein that plays a key role in activation of nuclear factor κ-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 αvβ3-selective cyclic, dimeric peptide 68Ga-DOTA-E[c(RGDfK)]2, where E[c(RGDfk)]2 = glutamic acid-[cyclo(arginyl-glycyl-aspartic 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 = 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 68Ga-DOTA-E[c(RGDfK)]2 After the last 68Ga-DOTA-E[c(RGDfK)]2 peptide PET/CT, tumors were cut into cryosections for autoradiography, histology, and immunohistochemistry. Results: The ex vivo uptake of 68Ga-DOTA-E[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 68Ga-DOTA-E[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.

Shank-associated RH domain-interacting protein expression is upregulated in entodermal and mesodermal cancer or downregulated in ectodermal malignancy.

Shank-associated RH domain-interacting protein (SHARPIN) is a type of linear ubiquitin chain-associated protein, which serves an important role in cell proliferation, apoptosis, organ development, immune and inflammatory reaction, initiation and development of malignant tumors. To evaluate SHARPIN expression in multiple malignant tumors derived from different germ layers, 14 types of cancer and their corresponding normal tissues were examined. Immunohistochemistry was performed to semi-quantify SHARPIN expression in multiple malignant tumors, and immunofluorescence was performed to evaluate the subcellular localization of SHARPIN in various malignant tumors. All the recruited cancer and paracancer samples originated from entoderm and mesoderm showed an upregulated expression of SHARPIN, whereas the cancer types that originated from ectoderm exhibited a downregulated or loss of SHARPIN expression. SHARPIN was primarily localized in the cytoplasm of cells and exhibited a faint signal in the nucleus, with the exception for lung cancer and esophagus cancer, in which malignant cells had aberrantly large nuclei and limited cytoplasm, which produced a signal in the nucleus but not in the cytoplasm. Conclusively, SHARPIN expression was upregulated in entodermal and mesodermal cancer types, but downregulated in ectodermal cancer types, indicating SHARPIN could act as either oncogene or anti-oncogene in malignant tumors derived from different germ layers.

Innate immune adaptor MyD88 deficiency prevents skin inflammation in SHARPIN-deficient mice.

Mice deficient in SHANK-associated RH domain-interacting protein (SHARPIN), a component of the linear ubiquitin chain assembly complex (LUBAC), develop a spontaneous inflammatory disorder with pathologic hallmarks similar to atopic dermatitis and psoriasis in humans. Previous studies identified the crucial role of components of the TNF and IL-1 signaling pathways in the progression of disease in SHARPIN-deficient mice. However, an innate immuneadaptor or sensor that relates to the disease progression has remained unknown. In this study, we found that the genetic ablation of myeloid differentiation primary response 88 (MyD88) completely rescued skin inflammation in SHARPIN-deficient (Sharpincpdm) mice. Systemic inflammation and immune cell dysregulation were partially rescued. Fibroblasts derived from SharpincpdmMyd88-⁄- mice failed to provide protection against TNF-induced cell death. SharpincpdmMyd88-⁄- mice had reduced TNF production in their skin. Furthermore, depletion of the microbiota through the oral administration of antibiotics(ABX) partially rescued both the skin inflammation and systemic inflammation, demonstrating a role for the gut microbiota in SHARPIN-deficient mice. Our findings suggest a detrimental role for the innate immune adaptor MyD88 in instigating skin inflammation in Sharpincpdm mice.

Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice.

SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-κB activity. SHARPIN-deficient (Sharpincpdm/cpdm) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpincpdm/cpdm mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpincpdm/cpdm phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1-/- Sharpincpdm/cpdm double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpincpdm/cpdm skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpincpdm/cpdm mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpincpdm/cpdm mice.

Emerging role of SHARPIN in hepatocellular carcinoma progression

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the leading cause of cancer-related death, especially in less economically developed regions. Despite recent progress in the diagnosis and therapy of HCC, the long-term survival rate of HCC patients is unacceptably low, in part due to the frequent development of vascular invasion or distant metastasis. The cellular functions of shank-associated RH domain-interacting protein (SHARPIN, also known as SIPL1) include the regulation of inflammation, apoptosis, immune signaling, and cell motility. SHARPIN is up-regulated in various types of cancers including HCC and has been implicated in the genesis and progression of malignant tumors, but its exact role in tumorigenesis is largely unknown. Here we present evidence supporting a role for SHARPIN in HCC invasion and progression. We also discuss the potential of SHARPIN and related genes as therapeutic targets for this currently incurable cancer.

A novel SHARPIN-PRMT5-H3R2me1 axis is essential for lung cancer cell invasion.

SHARPIN (Shank-associated RH domain interacting protein) is the main component of the linear ubiquitin chain activation complex (LUBAC). SHARPIN is involved in regulating inflammation and cancer progression. However, whether SHARPIN plays an important role in lung cancer metastasis and the potential underlying mechanism are still unknown. Here, for the first time, we reported that SHARPIN expression is closely related to lung cancer progression. Moreover, SHARPIN plays a central role in controlling lung cancer cell metastasis. Mechanistic studies further revealed that PRMT5 (Protein arginine methyltransferase 5), responsible for catalyzing arginine methylation on histones, is a novel cofactor of SHARPIN. This finding provides the basis for further study of the crosstalk between protein ubiquitination and histone methylation. We further found that SHARPIN-PRMT5 is essential for the monomethylation of histones of chromatins at key metastasis-related genes, defining a new mechanism regulating cancer invasion. A novel MLL complex (ASH2 and WDR5) was implied in the link between histone arginine2 monomethylation (H3R2me1) and histone lysine4 trimethylation (H3K4me3) for the activation of metastasis-related genes. These novel findings establish a new epigenetic paradigm in which SHARPIN-PRMT5 has distinct roles in orchestrating chromatin environments for cancer-related genes via integrating signaling between H3R2me1 and H3K4me3.

Lack of interaction between NEMO and SHARPIN impairs linear ubiquitination and NF-κB activation and leads to incontinentia pigmenti

Incontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding nuclear factor κB (NF-κB) essential modulator (NEMO; the regulatory subunit of the IκB kinase [IKK] complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here we describe a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO. We sought to identify the mechanism or mechanisms by which the truncated NEMO protein inhibits the NF-κB signaling pathway. We sequenced the IKBKG gene in patients with IP and performed complementation and transactivation assays in NEMO-deficient cells. We also used immunoprecipitation assays, immunoblotting, and an in situ proximity ligation assay to characterize the truncated NEMO protein interactions with IKK-α, IKK-β, TNF receptor-associated factor 6, TNF receptor-associated factor 2, receptor-interacting protein 1, Hemo-oxidized iron regulatory protein 2ligase 1 (HOIL-1), HOIL-1-interacting protein, and SHANK-associated RH domain-interacting protein. Lastly, we assessed NEMO linear ubiquitination usingimmunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1β. We identified a novel splice mutation in IKBKG (c.518+2T>G, resulting in an in-frame deletion: p.DelQ134_R256). The mutant NEMO lacked part of the CC1 coiled-coil and HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in activation of NF-κB signaling. The IL-1β-induced formation of NEMO-containing structures was impaired in fibroblasts from patients with IP carrying the truncated NEMO form (as also observed in HOIL-1-/- cells). The truncated NEMO interaction with SHANK-associated RH domain-interacting protein was impaired in a male fetus with IP, leading to defective linear ubiquitination. We identified a hitherto unreported disease mechanism (defective linear ubiquitination) in patients with IP.

Reduced SHARPIN and LUBAC Formation May Contribute to CCl₄- or Acetaminophen-Induced Liver Cirrhosis in Mice.

Linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on nuclear factor-κB (NF-κB) essential modulator (NEMO) and induces NF-κB pathway activation. SHARPIN expression and LUBAC formation were significantly reduced in the livers of mice 24 h after the injection of either carbon tetrachloride (CCl4) or acetaminophen (APAP), both of which produced the fulminant hepatitis phenotype. To elucidate its pathological significance, hepatic SHARPIN expression was suppressed in mice by injecting shRNA adenovirus via the tail vein. Seven days after this transduction, without additional inflammatory stimuli, substantial inflammation and fibrosis with enhanced hepatocyte apoptosis occurred in the livers. A similar but more severe phenotype was observed with suppression of HOIP, which is responsible for the E3 ligase activity of LUBAC. Furthermore, in good agreement with these in vivo results, transduction of Hepa1-6 hepatoma cells with SHARPIN, HOIL-1L, or HOIP shRNA adenovirus induced apoptosis of these cells in response to tumor necrosis factor-α (TNFα) stimulation. Thus, LUBAC is essential for the survival of hepatocytes, and it is likely that reduction of LUBAC is a factor promoting hepatocyte death in addition to the direct effect of drug toxicity.