Introduction

SHARPIN or the SHANK- associated RH domain-interacting protein or Shank-interacting protein-like 1 or SIPL1 is a multifunctional protein expressed in almost all cell types, conserved in all vertebrates (SHARPIN SHANK associated RH domain interactor [Homo sapiens (human)]—Gene—NCBI n.d.). SHARPIN has been initially identified as a synaptic protein (Lim et al. 2001) that directly interacts with SHANK (SH3 (Src homology 3) and multiple ankyrin repeat domains protein) and is highly enriched in the postsynaptic density (PSD) of excitatory neurons in the brain. The Shank family of proteins (also termed CortBP, Pro- SAP, or Synamon) contains multiple domains for protein–protein interactions, including ankyrin repeats, SH3 domain, PDZ (PSD-95/Dlg/ZO-1) domain, SAM (sterile alpha motif) domain, and an extensive proline-rich region (Boeckers et al. 2002), which appears to be regulated by alternative splicing (Lim et al. 1999). SHARPIN directly interacts with the ankyrin repeats of Shank and forms a complex with Shank in heterologous cells and brain (Park et al. 2003). Apprehensively, the possibility of SHARPIN playing a role in neuronal development and probably, even in synaptic plasticity and memory formation still remains unknown even after the identification of the protein in neurons in 2001.

Later, most of the studies in SHARPIN were concentrated on its role in inflammation and cancer after Seymour et al. identified the spontaneous mutation in SHARPIN causing chronic proliferative dermatitis (cpdm) in mice (Seymour et al. 2007) and Jung et al. identified the upregulation of SHARPIN in multiple types of cancer (Jung et al. 2010), respectively. SHARPIN was later found to regulate mechanisms that culminate in inflammation and apoptosis, thus confirming its role in immunity and cancer. The protein has been found to be a part of the linear ubiquitin chain assembly complex (LUBAC), which transfers linear ubiquitin chains to proteins targeting them for proteosomal degradation. Several other molecular mechanisms have been identified, where SHARPIN plays a significant role, both as a LUBAC-dependent and LUBAC-independent protein. Till now, the primary role of SHARPIN has been linked in association with HOIL-1 and HOIP, the other two components of the LUBAC complex (Ikeda et al. 2011). The Ubiquitin-like domain (UBL) at the C-terminal of SHARPIN is required for transferring ubiquitin and the Npl4-zinc finger domain (NZF) is required for the formation of LUBAC complex by associating with HOIL-1 (Stieglitz et al. 2012). The pleckstrin homology domain (PH) at the N-terminal of SHARPIN is essential for protein dimerization and is proposed to be required for LUBAC-independent functions, which include the role as a proto-oncogene in tumor progression (Jk et al. 2011).

In this context, the protein, initially identified as a postsynaptic protein, was well analyzed for two decades on its structural aspects, its function as an upstream regulator of the most explored transcription factor—NF-κB, and its role in immunity and in cancer. However, it still remains a question how this protein could function as a postsynaptic protein. The SHARPIN mutant mouse models showing no noticeable neurological defects, however, raise more questions than answers. Does this mean that SHARPIN has no role in a human brain or in the entire nervous system? New studies identifying role for SHARPIN in neurodegenerative diseases like Alzheimer’s disease and Amyotrophic lateral sclerosis (Asanomi et al. 2019; Krishnan et al. 2020; Nakayama et al. 2020) suggest an inevitable role for the protein in normal neuronal functioning. This review first summarizes the role of SHARPIN in a variety of normal cellular functioning and diseases and then contemplates on the not much known association of SHARPIN, amyloid-beta degradation, and Alzheimer’s disease, presumably via NF-κB signal modulators.

As a Member of LUBAC Complex

The primary role of SHARPIN has been identified to be associated with the LUBAC, where it interacts with two other components of the complex: the HOIL-1L (also known as RBCK1) and HOIP (also known as RNF31) (Gerlach et al. 2011; Tokunaga et al. 2009). HOIP and HOIL-1L are members of the RBR (RING-in-between-RING) E3 ubiquitin ligase family, where they are involved in the transfer of ubiquitin (Ub) molecules through two steps: first the Ub molecule is transferred to the catalytic cysteine in the RING2 of the E3 and then to the substrate (Kelsall et al. 2019; Morreale and Walden 2016). HOIP is the catalytic component and HOIL-1L is required for the formation and stability of the LUBAC complex (Peltzer et al. 2018). SHARPIN, as a component of the LUBAC, mediates TNF-α, CD-40 and other inflammatory stimuli-mediated activation of NF-κB by adding linear polyubiquitin chains on NF-κB Essential Modulator (NEMO), hence contributing to inflammatory mechanisms and the absence of SHARPIN leads to chronic proliferative dermatitis phenotype (SHARPINcpdm) in mice (Gerlach et al. 2011; Iwai 2011; Rickard et al. 2014; Tokunaga et al. 2011).

The interaction between NEMO and SHARPIN through the LUBAC complex thus regulating transcription factors like NF-κB and AP1 in response to TLR signaling was identified using systems analysis in bone marrow-derived macrophages isolated from SHARPIN-deficient or SHARPINcpdm mouse and confirmed using ‘NEMO harboring the panr2 mutation,’ the mutation that alters its interaction with SHARPIN (De et al. 2011). Genetic mutations in NEMO affecting its interaction with SHARPIN also altered NF-κB activity (Bal et al. 2017). The deletion of SHARPIN reduced the activity of LUBAC and hence NF-κB activation thereby confirming the role of SHARPIN as the third and important component of LUBAC complex. LUBAC-induced polyubiquitin-mediated proteosomal degradation of NEMO leads to the phosphorylation of IKKα/β (IκB Kinase), thereby phosphorylating and releasing the inhibitor of κB (IκB) allowing the translocation of active NF-κB into the nucleus for transcriptionally regulating the genes required for antiapoptosis and inflammation (Ikeda et al. 2011; Sieber et al. 2012; Tokunaga et al. 2011) as represented in Fig. 1. The inhibition of TNF-α expression rendered SHARPIN mutation-associated phenotypic defects (Gerlach et al. 2011), which revealed the molecular mechanisms that activate SHARPIN and the downstream signaling targets of the protein. In short, SHARPIN deficiency sensitizes the cells or organs associated with inflammatory stimuli like TNF-α (Kumari et al. 2014; Rickard et al. 2014), leading to exacerbated inflammatory activation, apoptosis, and necroptosis. Similar disease phenotype as seen in SHARPINcpdm mice has been identified in humans (X-linked hyper-IgM syndrome and hypohydrotic ectodermal dysplasia), with genetic mutation in NEMO (Tokunaga et al. 2011). A single case of HOIP mutation has been also reported, with similar pathologic features of autoinflammation and immune-deficiency in an 8-year-old girl (Oda et al. 2019). With conclusive evidences on the role of SHARPIN in activating NF-κB signaling pathway, Liang et al. identified that SHARPIN associates with TRAF2 (tumor necrosis factor receptor-associated factors) to negatively regulate NF-κB pathway, thus concluding that whether SHARPIN activates or inhibits NF-κB depends on the upstream regulators, protein interactions, and genetic mutations on the functional domains and the cell type (Liang 2011a).

Fig. 1
figure 1

An illustration of the primary upstream and downstream signaling mechanisms involving SHARPIN, where inflammatory stimuli activates signaling mechanisms that converge to activate linear ubiquitination of NEMO by the LUBAC complex, formed by the association of HOIL-1, HOIP, and SHARPIN, thereby activating NF-κB-mediated gene transcription(Fujita et al. 2014). The structural domains of SHARPIN are depicted as an illustration

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SHARPIN has LUBAC-Independent Functions too

SHARPIN, independent of LUBAC, localizes to uropods of lymphocytes and binds to the lymphocyte-function-associated antigen-1 (LFA-1) thereby inhibiting intercellular adhesion molecule-1 (ICAM-1)-mediated lymphocyte adhesion hence promoting migration of lymphocyte cells (Pouwels et al. 2013). SHARPIN has also been found to maintain the function of platelets by binding to Integrin αIIbβ3, independently of LUBAC, and preventing integrin adhesive activity by arresting the αIIbβ3 in the resting state, thereby avoiding unnecessary aggregation of platelets and the formation of circulating thrombi (Kasirer-Friede et al. 2019). SHARPIN is also associated with the eyes absent 1 protein (Eya1), a protein necessary for the development of various organs in both vertebrates and invertebrates, and the knockdown or genetic mutations in SHARPIN leads to BOR (branchio-oto-renal) syndrome-like phenotype in mouse and zebrafish (Brophy et al. 2013; Landgraf et al. 2010). These few studies on the LUBAC-independent role of SHARPIN suggest a possible role of SHARPIN in neuronal functioning, as mentioned above, in association with SHANK protein. In short, SHARPIN is required for normal embryogenesis, development, differentiation and immunity, and an abnormal activation of SHARPIN, and hence the activation of both LUBAC-dependant and LUBAC-independent pathways leads to several defects ranging from embryonic lethality to terminal diseases like cancer, autoinflammation, cardiovascular diseases, and neurodegenerative diseases.

Role in Immunity and Inflammation

The first report on the role of SHARPIN in participating in immunity and inflammatory response was identified in a mouse carrying spontaneous genetic mutation in SHARPIN resulting in truncated protein expression, causing multi-organ inflammation, immune system dysregulation, defective secondary lymphoid organ development, and chronic dermatitis termed as chronic proliferative dermatitis (cpdm) (Potter et al. 2014; Renninger et al. 2010; Seymour et al. 2007), highlighting the direct involvement of SHARPIN in regulating the cellular and molecular components of inflammation in multiple pathways. Later, the over activation of NF-κB signaling was identified as the major cause for the phenotypic defects in the SHARPINcpdm mice. Further, the inhibition of NF-κB signaling using inhibitors was found to alleviate the symptoms in SHARPINcpdm mice. These findings hoist the initial signal that SHARPIN might be involved in regulating the NF-κB signalling mechanism (Liang 2011b; Liang et al. 2011).

Further research in exploring the role of SHARPIN in inflammation has identified that a single mutation in the protein could completely alter immune mechanisms (Wang et al. 2012). The work from our lab has shown that silencing of SHARPIN in macrophages altered immune response to proinflammatory stimuli like LPS by shifting the polarization of macrophage cells to the M2 antiinflammatory phenotype (Krishnan et al. 2020). The same mechanism has been elucidated by these cells in response to the pathological protein, Aβ, which is implicated in AD (Krishnan et al. 2020), highlighting the probable immune and inflammatory role regulated by SHARPIN in brain-resident microglial cells, the sister cells of circulating macrophages. SHARPIN-NEMO interaction is also necessary for negative regulation of antiviral response by macrophages through IFN (Interferon) pathway (Belgnaoui et al. 2012). TLR3-mediated host response against influenza A virus (IAV) is also regulated by LUBAC complex and SHARPIN deficiency leads to enhanced virus-associated cell death mimicking immunodeficiency and autoinflammation (Brazee et al. 2020; Zinngrebe et al. 2016). Lack of SHARPIN in mice (cpdm) during embryogenesis results in the formation of a rudimentary Peyer’s patches, the secondary lymphoid tissue located in the intestine of mammals providing adaptive immune response against intestinal pathogens (Seymour et al. 2013). SHARPIN plays a primary role in the development of atopic dermatitis through the activation of IL-33/ST2 signaling pathway (Tang et al. 2018). SHARPIN is also essential for the development, differentiation, and survival of regulatory T cells (Treg) (Okamura et al. 2016; Redecke et al. 2016; Teh et al. 2016) and SHARPIN-deficient mouse also showed an increase in the number and a severe dysregulation in the functioning of Treg cells (Park et al. 2016; Sasaki et al. 2019), proving the role of SHARPIN in both innate and adaptive immunity.

Later, linear ubiquitination by LUBAC was found to be required for the expression and activation of the NLRP3 inflammasome, a protein complex of NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 OR Nod-like receptor protein 3), the adaptor ASC (apoptosis-associated Speck-like protein containing a Caspase-recruitment domain), and pro-caspase-1, which functions as a cytosolic pathogen recognition receptor and activates inflammatory responses by the maturation and release of proinflammatory cytokines like IL-1β and IL-18 (Gurung et al. 2016; Rodgers et al. 2014). SHARPIN as a part of the LUBAC complex is exquisite for both canonical and non-canonical activation of NLRP3 inflammasome (Gurung et al. 2015), thus again proving as an essential part for inflammatory response to both external and internal danger stimuli. The activation of caspase-1 itself was also reported to be regulated by SHARPIN in response to enhanced inflammation (Nastase et al. 2016). In fact, the expression and activation of inflammatory caspases and IL-1 family of cytokines were highly upregulated in SHARPINcpdm mice, hence establishing the primary role of SHARPIN-mediated NLRP3 in regulating inflammatory cues and the resultant apoptotic pathways (Douglas et al. 2015). SHARPIN or HOIP deficiency also alternatively enhanced caspase-1 activity, inflammasome activation leading to cell death (Douglas and Saleh 2020). A different, yet remarkable alternate type of apoptosis called pyroptosis, in this context, is interesting to be noticed. Pyroptosis is a type of apoptosis in which NLRP3 plays the primary role, where the maturation of the IL-1 family of inflammatory cytokines initiates an inflammatory type of programmed cell death pathway, which will trigger the release of more inflammatory cytokines, thus activating the surrounding immune cells creating an inflammatory mileu (Man et al. 2017). Inflammasome-mediated pyroptosis has been well explored in many diseases (X. Chen et al. 2019a, b; Dubois et al. 2019; Qiu et al. 2017; Zeng et al. 2019) including neurodegenerative diseases like Parkinsonism (Wang et al. 2019), hence posing the need to explore the role of SHARPIN as a regulatory protein to prevent pyroptotic inflammatory damage, especially in the brain.

Modulating Apoptosis by SHARPIN: Lessons Learned

The role of SHARPIN as an upstream regulator of NF-κB activation itself proves its prerequisite role in regulating programmed cell death (Ikeda et al. 2011). The transcription factor NF-κB protects cells from apoptosis in response to proapoptotic stimuli by transcribing antiapoptotic genes like Bcl2, survivin, and livin required for survival of the cells (Dutta et al. 2006; Fan et al. 2008; Zhang et al. 2014). Hence, SHARPIN has an antiapoptotic role in response to apoptotic stimuli like inflammatory trigger by TNF (Sieber et al. 2012). Approximately 77% of the skin cells in SHARPIN-deficient mice were reported to be apoptotic, again stressing the role of SHARPIN as a protector against apoptosis (Liang and Sundberg 2011; Shimizu et al. 2016). This study also identified that keratinocyte apoptosis in SHARPIN-deficient mice was mediated through mitochondria-dependent pathway, initiated by mitochondrial damage and subsequent increase in the expression of BAX, caspase 3, and caspase 9 (Liang and Sundberg 2011; Potter et al. 2017).

Apart from this, the role of SHARPIN as a regulator of antiapoptotic pathway is evident from several disease conditions including cancer. Due to this role, SHARPIN is also regarded as a proto-oncogene with its antiapoptotic property, which is discussed in the next section of this review in detail.

Promoting Invasion and Metastasis in Cancer

The role of SHARPIN in promoting cancer cell survival and metastasis was first identified by Jung et al. in 2010 (Jung et al. 2010). A genome-wide analysis was performed between biopsies of tumor tissue and normal tissue and identified that SHARPIN was upregulated in many types of human cancer. Further, overexpressing SHARPIN in CHO cell lines enhanced their cancer-specific phenotypes, suggesting a superior role for SHARPIN in cancer biogenesis for the first time (Jung et al. 2010). Even though this study did not explore the mechanistic regulation of cancer progression by SHARPIN, later studies proved the role of SHARPIN in regulating NF-κB activation, which essentially could be the reason for enhanced cancer progression, since NF-κB transcriptionally regulates pro-survival genes. This is evident from the enhanced apoptosis in chronic proliferative dermatitis (cpdm) when SHARPIN was mutated causing multi-organ inflammation, which has been proven to be due to the dysregulation of NF-κB signaling in the absence of SHARPIN (Ikeda et al. 2011). Although the study showed the mechanism of regulating inflammatory phenotype in cpdm mice, this also could explain why SHARPIN overexpression is enhancing cancer phenotype-specific genes since many of the cancer pro-survival genes required for cell survival, metastasis, and angiogenesis are under the transcriptional regulation of NF-κB. SHARPIN was also shown to regulate keratinocyte apoptosis in cpdm mice through mitochondria-dependent pathway, initiated by mitochondrial damage then followed by an enhanced expression of BAX, caspase 3, and caspase 9 and reduced expression of BCl2 (Liang and Sundberg 2011), which could also contribute to cancer survival mechanisms induced by SHARPIN overexpression. TNF-α, one of the upstream regulator of SHARPIN-mediated NF-κB activation, could stimulate both pro- and antiapoptotic signaling pathways; however, the absence of SHARPIN causes exacerbated apoptosis in hepatocytes causing pre-mature liver damage (Sieber et al. 2012). All these studies indirectly underwrite to the cancer-promoting role of SHARPIN by regulating apoptosis in different cell types and tissues.

Later, studies confirmed that the involvement of SHARPIN in cancer progression is primarily through the NF-κB signaling pathway (Tomonaga et al. 2012). Activation of NF-κB by SHARPIN and LUBAC complex upregulated the expression of pro-survival genes and reduced the expression of proapoptotic markers which have been shown to promote progression and metastasis of prostate cancer, hepatocellular carcinoma, and mycosis fungoides (MF), the most common subtype of cutaneous T‐cell lymphomas (B. Chen et al. 2019a, b; Erdogan et al. 2017; Huang et al. 2017; J. Li et al. 2015a, b, p. 3; Tanaka et al. 2016; Yamamotoya et al. 2017; Zhang et al. 2014). The significant role of SHARPIN in breast cancer was first evident from the study by De Melo and Tang, where they identified an increased gene copy number and mRNA expression of SHARPIN in all the major subtypes of breast cancer, including estrogen receptor (ER) + , progesterone receptor (PR) + , HER2 + , and triple negative and that the mRNA levels correlated with the tumor grade and reduced patient survival rate (De Melo and Tang 2015) (Ojo et al. 2017). SHARPIN was also found to regulate Estrogen receptor α (ERα) and its downstream targets, indirectly regulating the receptor level by enhancing its stability by preventing polyubiquitination in ERα, thus contributing to breast cancer progression and low prognosis with a higher level of SHARPIN expression in ERα-positive patients (Zhuang et al. 2017). Subsequently, SHARPIN was identified as a metastasis gene and prognostic marker for breast cancer (Bii et al. 2015; Kharman-Biz et al. 2018; Ojo et al. 2018) and that p53/MDM2 complex is one of the major targets of SHARPIN-mediated signaling in cancer cells which facilitate p53 degradation by ubiquination (Yang et al. 2017). Alterations in the gene copy number of multiple genes were identified in malignant melanoma cells and among them, SHARPIN was identified as one with multiple copy numbers, which was associated with enhanced metastasis and invasiveness (Koroknai et al. 2016). SHARPIN mutation rate was also proven to be higher in non-melanoma skin cancer (NMSC) (Zheng et al. 2019) and SHARPIN expression was found to regulate the Ras-associated protein-1(Rap1) and downstream pathways enhancing the progression and metastasis of melanoma (Zhou et al. 2019).

Even though all these studies positively associate SHARPIN with tumor progression and cancer invasiveness, very few studies have shown contradicting results (Haris et al. 2014; HogenEsch et al. 2016). An elaborate study conducted on different cancer types originating from different germ layers indicated the most interesting fact on the role of SHARPIN in cancer, where the tumors originating from the endoderm and mesoderm show a higher SHARPIN expression and those arising from the ectoderm have a lower or downregulated expression of SHARPIN (Liang et al. 2018). Further, the study has shown differential expression of SHARPIN in the cytosol and nucleus and more importantly SHARPIN as a proto-oncogene and an anti-oncogene in different types of cancers. These contradictory findings could point to the multiple NF-κB-independent roles for SHARPIN in regulating apoptosis and yet unidentified cellular functions. This was evident from the later studies which prove different roles for SHARPIN apart from NF-κB activation.

SHARPIN was found as a negative regulator of Phosphatase and tensin homolog (PTEN), the tumor suppressor which inhibits tumor progression by downregulating PI3K-AKT pathway, thus inhibiting cell proliferation, adhesion, and migration (De Melo et al. 2014b, 2014a). Protein kinase C zeta (PKCζ), another tumor suppressor, is ubiquitinated and degraded via LUBAC activity, which was shown to enhance tumor size and adaptation to hypoxia in lung adenoma (Queisser et al. 2014). Another study revealed an interesting finding on the role of SHARPIN in associating with PRMT5 (Protein arginine methyltransferase 5), the protein responsible for catalyzing methylation on arginine residues on histone proteins. The SHARPIN-PRMT5 association was found to be essential for monomethylation of histone proteins on the chromatins with cancer metastasis-related genes, thus pointing out the epigenetic role for SHARPIN (Fu et al. 2017). This protein–protein interaction shows a strong role for SHARPIN independent of the LUBAC complex, where it also regulates the expression of transcription factors like SOX10 (Sry-related HMg-Box gene 10) and MITF (microphthalmia-associated transcription factor) (Tamiya et al. 2018). A recent study also identified SHARPIN association with another protein YAP (Yes-Associated Protein), where SHARPIN overexpression enhanced K48-polyubiquitination of YAP leading to its degradation, thus acting as an inhibitor of YAP and hence cancer progression (Zhang et al. 2019). Thus, SHARPIN performs extensive role in cell survival and cancer, either as LUBAC-dependent or as LUBAC-independent protein.

Roles in Cell Adhesion and Migration

A study by Xia et al. identified that SHARPIN-deficient mice models show a significant reduction in transcription factors required for bone development and differentiation like Runx2 and osterix, suggesting the role of the protein in all the aspects of bone metabolism including bone volume and bone mineral density (Xia et al. 2011). The role of SHARPIN in skeletal homeostasis was also evident since, apart from the inflammatory phenotype, loss in bone strength and dysregulation in osteoblast differentiation were identified as defects in SHARPINcpdm mice (Jeschke et al. 2015; McGowan et al. 2014). Xia et al. also pointed out a significant decrease in the levels of type I collagen and a noncollagenous protein hormone, osteocalcin (Xia et al. 2011). This study initially pointed out the role of SHARPIN in regulating the expression of extracellular matrix (ECM) proteins. The role of SHARPIN-mediated collagen regulation is also evident by the defects in collagen fiber assembly, function, and degradation causing ECM stiffness and reduced mammary ductal outgrowth in SHARPINcpdm mice (Peuhu et al. 2016).

An RNAi screening has found SHARPIN as an important negative regulator of β1-integrin activity (Jk et al. 2011). Integrins are heterodimeric transmembrane proteins with α and β subunits that help in forming cell-to-cell and cell-to-ECM adhesion. SHARPIN, independently of LUBAC, binds directly to the cytoplasmic region of integrin-α subunits, thereby hindering its binding to the β1-integrin activators, talin and kindlins. This mechanism of β1-integrin inactivation by SHARPIN was observed in tumor cells thereby regulating tumor cell migration and also in SHARPINcpdm mouse enhancing epidermal hyperproliferation (Jk et al. 2011; Peuhu et al. 2017; Siitonen et al. 2019). Later it was confirmed that SHARPIN binds to integrin via the same UBL domain required for its binding with HOIP forming the LUBAC complex. SHARPIN also binds with kindlin-1, which enhanced the binding efficiency of SHARPIN with integrin β1 (Gao et al. 2019, p. 1). The inhibition of SHARPIN-Integrin interaction specifically activated β1-integrin without hindering its LUBAC-dependent function and the inhibition of SHARPIN-HOIP interaction affected NF-κB activity only, without activating β1-integrin, which confirmed that the role of SHARPIN as β1-integrin inhibitor is LUBAC independent (De Franceschi et al. 2015). In fact, the SHANK protein, especially SHANK 1 and 3, itself has actin-regulating function and acts as an inhibitor of integrin activation by binding and sequestering Rap1 and R-Ras, the activators of integrin, thus hindering the interaction between integrin and the activators (Lilja et al. 2017). This LUBAC-independent SHARPIN-integrin αIIbβ3 interaction is also important in inflammatory mechanisms by platelets for fibrinogen-dependent platelet aggregation, MHC class I presentation, and proinflammatory activation (Kasirer-Friede et al. 2019).

SHARPIN is also important for cell migration by regulating lamellipodium formation. Actin-related protein 2/3 (Arp2/3) complex is required for catalyzing the formation of actin filaments that activate the formation of lamellipodium by initiating a protrusion in the cell due to a meshwork of actin formation (Wu et al. 2012). An interactome study identified that SHARPIN, independent of LUBAC, directly interacts with Arp2/3 through the UBL domain and promotes lamellipodium formation thus helping in cell migration (Khan et al. 2017).

SHARPIN: A Double-Edged Sword in Alzheimer’s Disease

SHARPIN, with a huge impact in many diseases, has been recently investigated to be involved in one of the most devastative neurodegenerative disease, Alzheimer’s disease. In 2018, a genome-wide association study (GWAS) in Alzheimer’s disease predicted the involvement of novel genes that are possible risk factors for AD and SHARPIN was one among them (Lancour et al. 2018). In this study, the authors constructed a network of direct and indirect protein–protein interactions and ranked the genes based on their proximity to 60 robust AD (RAD) genes. Using bioinformatics tools, they identified three genes, SHARPIN, CR2 (complement receptor type 2), and PTPN2 (protein tyrosine phosphatase non-receptor type 2), which are involved in the regulation of inflammatory mechanisms and microglial function, found to be associated with AD. Recently, Asanomi et al. identified a rare functional variant of SHARPIN as a genetic risk factor for late-onset sporadic form of Alzheimer’s disease. The study analyzed genetic mutations in late-onset AD patients without the Apolipoprotein Eε4 risk allele (the most prominent genetic risk factor of late-onset AD) using whole exome sequencing analysis and found that the presence of this genetic mutation (rs572750141, NM_030974.3:p.Gly186Arg) leads to the expression of a functional variant of SHARPIN which on preliminary analysis was shown to have affected NF-κB activity by reducing its nuclear translocation thereby attenuating inflammatory response in AD. The mutation was found to enhance the risk for AD by 6.1 times, higher than the known AD risk factor TREM2 (Asanomi et al. 2019). An online preprint version of another study identified that a nonsynonymous mutation (rs34173062: p.Ser17Phe) in SHARPIN, affecting dimerization and scaffolding of SHARPIN, correlates with reduced cortical thickness in entorhinal region and enhanced amygdalar atrophy, the most vulnerable area in AD (Soheili-Nezhad et al. 2019). More studies need to be done to identify the signaling mechanisms regulated by SHARPIN in contributing to brain development and neuronal maintenance. Studies from our lab analyzed the functional role of SHARPIN in AD and we have identified novel roles for SHARPIN in regulating amyloid-beta (Aβ) phagocytosis and Aβ-mediated inflammatory mechanisms in macrophages promoting neurodegeneration in AD (Krishnan et al. 2020). We found that SHARPIN protein acts as a double-edged sword in AD scenario, where a reduced expression causes defective Aβ clearance leading to pathogenic Aβ accumulation, whereas an increased expression contributes to enhanced Aβ-mediated inflammatory mechanisms causing neuronal death. In either way, SHARPIN contributes to neuronal death and hence maintenance of an optimum level of SHARPIN is prerequisite for maintaining proper immune response to Aβ in Alzheimer’s disease (Krishnan et al. 2020). In this context, Gliotoxin was earlier identified as an inhibitor of LUBAC complex and hence enhances NF-κB activity, by binding and inhibiting the catalytic domain of LUBAC, HOIP (Sakamoto et al. 2015). The same toxin was found to inhibit macrophage function (Schlam et al. 2016) stating the significant role played by the LUBAC complex in modulating macrophage function. The SHARPIN expression was also found to be higher in the early stages of AD patients in the blood-derived macrophages and correlated with the circulating Aβ levels, suggesting a prominent functional role for the protein in promoting AD (Krishnan et al. 2020). Since the recent finding could prove the genetic and functional role of SHARPIN in AD, lots of research needs to be done in the field of AD to explore the mechanisms regulated by SHARPIN in protecting neuronal health and promoting neurodegeneration. SHARPIN, as a LUBAC component and functioning as an NF-κB modulator, the role of the protein and of LUBAC complex as such in contributing to AD-associated neurodegeneration, is worth exploring. At this point, it is remarkable to note that all the mechanisms involved in Amyloid-β Clearance and Degradation (ABCD) pathways are under the transcriptional regulation of NF-κB, which is activated by LUBAC-dependent SHARPIN-mediated signaling mechanisms. The expression of majority of the enzymes required for Aβ degradation like matrix metalloproteinases (MMPs) and cathepsins is under the transcriptional control of NF-κB transcription factor, thus pointing to its role in maintaining the physiological level of Aβ in the brain (Baranello et al. 2015). Interestingly, the amyloid-precursor protein (APP), the parental protein from which Aβ is produced, and the beta site APP cleaving enzyme (BACE), responsible for the mismetabolism of APP producing Aβ (Reitz 2012), are also under the transcriptional control of NF-κB and hence possibly regulated by SHARPIN/LUBAC, thus ascertaining the role of SHARPIN in the progression of AD. Two major proteins that regulate the shuttling of Aβ through the blood–brain barrier; RAGE—regulating the uptake of Aβ from the blood to brain and LRP1—regulating the clearance of Aβ into the circulation from brain plays very important role in the maintenance of the physiological level of Aβ in the brain, preventing the development of AD. It is interesting to note that the level of RAGE is upregulated and LRP is downregulated or modified in AD, thus increasing Aβ accumulation in the brain (Deane et al. 2009, 2004). Both these proteins, with opposite functions, are also under the transcriptional regulation of NF-κB, further strengthening the double-sided role of SHARPIN in Aβ metabolism and thus in the pathogenesis of Alzheimer’s disease. Hence, the functional implications of the genetic variant of SHARPIN need to be explored in deep in promoting neurodegeneration, specifically AD. The title of this review is made to implicate the not much known association of SHARPIN, Aβ metabolism, and Alzheimer’s disease, via NF-κB signal modulators.

A recent study also identified SHARPIN and HOIP colocalized with the linear ubiquitin chains in the neuronal cytoplasmic inclusions (NCIs) associated with another neurodegenerative disease, and amyotrophic lateral sclerosis (ALS) along with the other well-known protein depositions in ALS, including TAR DNA-binding protein (TDP-43). The active NF-κB component p65, which is released as a result of the SHARPIN-mediated LUBAC activation, was also found to be associated with the NCIs, suggesting that the SHARPIN-mediated L-Ub of NCIs may play a functional role in inducing autophagic clearance of NCIs, neuroinflammation, and neurodegeneration in ALS (Nakayama et al. 2020). TDP-43 was also reported to be colocalized with NF-κB subunit p65 and many studies suggested that the inhibition of NF-κB reduced ALS-associated symptoms in TDP-43 mice models pointing to a role for NF-κB associated signaling pathway as a therapeutic target for ALS treatment (Dutta et al. 2020; Patel et al. 2015; Swarup et al. 2011). Several proteins that are associated with ALS like TDP-43 fused in sarcoma (FUS), p62/SQSTM1, and Ubiquilin-2 (UBQLN2) were found to modulate NF-κB pathway (Picher-Martel et al. 2015). These studies provide a very strong and significant role for SHARPIN, an upstream regulator of NF-κB and thereby the LUBAC complex in neurodegenerative diseases, even though elaborate studies need to be conducted concentrating on its specific and mechanistic role played in the pathogenesis, progression, and may be as a predictive biomarker and/or a drug target for neurodegenerative diseases, especially Alzheimer’s disease. Further, it is interesting to note that even though the protein was initially identified as a neuronal postsynaptic protein, its role in maintaining neuronal function, synaptic plasticity, and memory formation remains elusive. Even though no evident neuronal defect was observed in mouse models carrying mutations resulting in truncated SHARPIN expression (Seymour et al. 2007), the role of this protein in human neuronal functioning needs to be explored (Fig 2)

Fig. 2
figure 2

SHARPIN as an important mediator for regulating the physiological levels of Aβ. SHARPIN activated by various stimuli might be involved in the generation of Aβ from APP by an increased expression of APP and BACE1, which increases Aβ accumulation and hence AD risk. On the contrary, SHARPIN regulates immune-mediated clearance of Aβ and possibly enzyme- and blood–brain barrier-mediated clearance of Aβ, thereby reducing Aβ accumulation and hence a reduced risk for AD. This proposes the importance of SHARPIN in AD pathogenesis

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Therapeutic Approaches

On a therapeutic aspect, the inhibition or activation of SHARPIN expression to treat or prevent SHARPIN deficiency or inflammatory-associated diseases is of less hope since the protein is ubiquitously expressed and the complete role of the protein in development, metabolism, and disease still remains to be understood. As the maintenance of NF-κB signaling at an optimal level is essential to prevent early apoptosis and inflammatory activities of the cells, maintaining the optimum levels of SHARPIN, to regulate NF-κB and all the other molecular pathways regulated by SHARPIN, is tricky. On the other hand, the universal role played by the protein in many types of cancers, inflammatory, and cardiovascular diseases and even in neurodegenerative diseases hoists the need to target SHARPIN or its regulatory pathways extremely essential. Several studies in mouse models has identified possible targets to tally the effects of SHARPIN mutation-induced inflammatory damage; however, the unexplored areas of SHARPIN involvement in human beings, specifically in brain development, suggest a potential risk in targeting SHARPIN in these disease conditions.

Crossing SHARPINcpdm with kinase-dead RIP1(Ripk1(K45A)) mice has rescued all the pathologic features of the cpdm mice and also the direct inhibition of RIPK1 prevented skin inflammation in these disease models (Berger et al. 2014; Patel et al. 2020; Webster et al. 2020). These studies provide hopes for RIPK1 as a therapeutic target for TNF-mediated inflammatory diseases, which can be further extended as a therapeutic target for SHARPIN and LUBAC-mediated diseases. RIPK1 phosphorylation at Ser25 by TNF-mediated signaling was defective in SHARPINcpdm cells and restoration or mimicking the phosphorylation rescued the cells from inflammatory damage (Dondelinger et al. 2019). In this context, Optineurin (OPTN), an ALS-associated protein in which the loss of function mutation leads to neurodegeneration, is recruited by the LUBAC complex, which in turn activates NF-κB leading to inflammation and apoptosis in ALS (Nakazawa et al. 2016; Oikawa et al. 2020a). OPTN was also found to regulate RIPK1 expression by targeting RIPK1 for proteosomal degradation and the loss of function mutation in OPTN leads to overexpression of RIPK1, thus causing enhanced inflammation-mediated axonal degeneration and dysmyelination in the spinal cord. Further, the inhibition of RIPK1 rescued the cells from inflammatory damage (Ito et al. 2016), thus pointing an indirect role of SHARPIN and LUBAC in ALS neurodegeneration and RIPK1 inhibition as a treatment modality for inflammation-mediated neuronal damage in ALS. Another study suggested a role for myeloid differentiation primary response 88 (MyD88) in regulating SHARPIN deficiency-mediated disease pathogenesis, where genetic ablation of MyD88 rescued SHARPINcpdm mice from skin inflammation although partial recovery was seen in systemic inflammation and immune cell dysregulation (Sharma et al. 2019). The inability to form the LUBAC complex when HOIP is post-translationally modified by ubiquitination at Lys1056 (Bowman et al. 2015) can also render inflammatory activation. Katsuya et al. identified a series of small molecule inhibitors named as HOIPINs (HOIP INhibitors) that could act as inhibitors of NF-κB signaling by inhibiting LUBAC activity (Katsuya et al. 2019; Oikawa et al. 2020b). Synthetic stapled α-helical peptides (peptides with secondary α-helix stabilized by hydrophobic bridge) against HOIP have also been identified as possible therapeutic strategy to inhibit LUBAC function (Aguilar-Alonso et al. 2018). Fujita et al. identified an α-helical stapled peptide mimicking the LUBAC-tethering motifs (LTM) of SHARPIN, thus acting as an inhibitor of LUBAC by disrupting the interaction between SHARPIN and HOIL-1L (Fujita et al. 2018). Several other HOIP-mediated LUBAC inhibitors have been identified in vitro and in vivo till date (Johansson et al. 2019). Whether a precise targeting of a specific function of SHARPIN, either the LUBAC-dependent functions without hindering the LUBAC-independent functions or vice-versa, will be possible is an important question that should be raised at this point.

Negative regulation of NF-κB signaling, when SHARPIN-mediated checkpoint is dysregulated, serves as another alternative. In this regard, the protein A20 (TNFAIP3) inhibits NF-κB activation in response to TNF and TLR-mediated signaling, which is proved to be another therapeutic approach (Verhelst et al. 2012). The Zinc Finger (ZF) 7 domain of A20 has the potential to inhibit LUBAC-mediated NF-κB activation by binding to the linear polyubiquitin chains, thereby inhibiting the interaction between LUBAC and NEMO (Verhelst et al. 2012). A20 has also been suggested as a tumor suppressor (Schmitz et al. 2009), thus pointing A20 as a potential therapeutic target in TNF or LUBAC-mediated inflammatory diseases and/or cancer. Another protein called ABIN-1 (A20 binding and inhibitor of NF-κB), an antagonist of LUBAC, when overexpressed has also been identified to significantly reduce TNF or TRAIL (TNF-related apoptosis-inducing ligand)-mediated NF-κB activation (Dorn et al. 2018, p. 293). Inhibition of calpain activation was found to inhibit NF-κB-mediated inflammation by decreasing the expression of many upstream regulators including SHARPIN, thus preventing chronic myocardial ischemia (Potz et al. 2017). The potential use of these therapeutic strategies in preventing neurodegenerative diseases like AD, possibly by maintaining Aβ metabolism and Aβ-mediated inflammatory damage, should be hypothesized.

Perspectives: Balancing act of SHARPIN in Neurodegenerative Diseases

An interesting finding identified that Parkin-coregulated gene (PACRG) could act as a substitute to SHARPIN, by binding to LUBAC complex in the absence of SHARPIN and activating NF-κB translocation in response to TNF-α signaling (Meschede et al. 2020). This would be of great interest in this context since PACRG was reported to suppress unfolded Pael receptor (Parkin substrate)-induced cell death, forming Lewy bodies and protecting neurodegeneration of dopaminergic neurons in Parkinson's disease, even though knockout studies failed to see any behavioral deficits in mice (Stephenson et al. 2018). The possibility of SHARPIN regulating Aβ metabolism, as previously mentioned in this review, and hence in Alzheimer’s disease pathogenesis, is also considerable. SHANK family of proteins are major scaffolding proteins in the postsynaptic terminal and genetic mutations in the genes have been associated with autism spectrum disorders, which prove their eminent role in neuronal development and synaptic connections (Monteiro and Feng 2017). An interesting study by Mameza et al. identified that genetic mutations in SHANK3 ankyrin repeat region (ARR), the domain required for its interaction with SHARPIN, and mutations in Shank/ProSAP N terminus (SPN) region enhance its binding efficiency to SHARPIN. A L68P genetic mutation identified in an autistic patient makes the ARR domain permanently accessible to its ligands including SHARPIN (Mameza et al. 2013), which in turn suggests that enhanced binding to the partners of SHANK might contribute to neurodevelopmental disorders, again stressing the role of SHARPIN as an important protein in nervous system development. In this context, the protein SHARPIN, identified initially as a protein associated with the postsynaptic protein SHANK, with emerging roles in neurodegenerative diseases, needs to be explored in terms of its LUBAC-dependent and independent roles in neuronal functioning and neuronal development. The unknown role of SHARPIN in the postsynaptic terminal, the physiological role of Aβ as an enhancer of long-term potentiation (LTP) and modulation of synaptic transmission (Brothers et al. 2018), and the possible direct or indirect interaction between Aβ and SHARPIN may further suggest a potential direct role of SHARPIN in neuronal functioning and AD pathogenesis. Our study has indirectly shown that an optimum level of SHARPIN is required to regulate the physiological level of Aβ degradation and inflammatory mechanisms by peripheral macrophages in AD. An alteration in the level of SHARPIN expression either increases Aβ levels or enhances inflammation-mediated neuronal death, both would be catastrophic in Alzheimer’s disease (Krishnan et al. 2020). SHARPIN is also responsible for inflammation-mediated neuronal damage in the facial nucleus of the brain stem in response to herpes simplex virus type 1 (HSV-1) causing facial paralysis or Bell’s palsy (Y. Li et al. 2015a, b). In conclusion, SHARPIN’s role as an inflammatory activator may also play an important role in synaptic maintenance in both central and peripheral nervous system, by regulating inflammatory activation of microglia and astrocytes thus preventing inflammation-mediated synaptic pruning and neuronal damage in many diseases including neurodegenerative diseases. Nevertheless, it would be worthwhile to study the complex relationship of SHARPIN, inflammatory mediators, and Aβ metabolism in AD.