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Ubiquitination is a reversible post-translational modification that regulates function of conjugated proteins by decorating with ubiquitin chains—polymer of ubiquitin—in most cases. The discovery of linear ubiquitin chains and the linear ubiquitin chain assembly complex (LUBAC) ubiquitin ligase complex can be considered as paradigm shift in the ubiquitin research because the linear ubiquitin chain is generated via the N-terminal Met of ubiquitin, although the other ubiquitin chains are generated via one of seven Lys residues in ubiquitin. Moreover, ubiquitination is distributed throughout eukaryotic kingdoms; however, no linear ubiquitination could be found in lower eukaryotes including yeasts. Although the involvement of ubiquitination in proteolysis is well-documented, linear ubiquitination plays crucial roles in immune signaling and cell death regulation. Moreover, dysregulation of LUBAC-mediated linear ubiquitination underlies various human diseases including autoinflammation and cancer. Here, I introduce how linear ubiquitination was discovered and outline a brief history of linear ubiquitination research.
- chronic proliferative dermatitis in mice
- heme-oxidized IRP2 ubiquitin ligase 1
- HOIL-1L interaction protein
- IκB kinase complex
- iron regulatory protein 2
- inhibitor of κB
- LUBAC-tethering motifs
- linear ubiquitin chain assembly complex
- NF-κB essential modifier
- nuclear factor-κB
- Npl4 zinc finger
- OTU deubiquitinase with linear linkage specificity
- RING-in between RING-RING
- SHANK-associated RH domain interactor
Although proteins are functional molecules, they do not always exert their functions. Post-translational modifications, including phosphorylation and ubiquitination, regulate protein function through covalent conjugation of specific molecules to amino acid residues of proteins. Ubiquitin is a highly conserved proteinous post-translational modifier that is widely distributed throughout eukaryotic kingdoms. I first became acquainted with ubiquitin when I worked as a postdoctoral fellow in the laboratory of Drs Tracey A. Rouault and Richard D. Klausner at NICHD in 1994. I applied for a postdoctoral position in Dr Klausner's laboratory because he performed ground-breaking work on T-cell receptor signaling []. Because my graduate work focused on autoimmune diseases, I was interested in joining his laboratory. Based on the recommendation of immunology experts from Kyoto University, including Dr Tasuku Honjo, Dr Klausner allowed me to join his laboratory. However, Dr Klausner stopped working on T-cell receptor signaling when I joined, and I became involved in another project, namely regulation of cellular iron metabolism. During my stay at NICHD, I found that the function of iron regulatory protein 2 (IRP2), one of the main regulators of iron metabolism, is controlled by iron-dependent degradation of the protein by the 26S proteasome []. This is how I became acquainted with ubiquitin.
Engagement with the ubiquitin–proteolytic system
The ubiquitin system was discovered as part of the energy-dependent protein degradation pathway termed the ubiquitin–proteasome system in 1978 by Drs Aaron Ciechanover and Avram Hershko []. In this system, ubiquitin conjugation functions as a recognition signal for the 26S proteasome and targets the conjugated proteins for degradation. Although the involvement of the ubiquitin system in cell cycle control was demonstrated in 1984 [], the physiological role of the ubiquitin–proteasome pathway remained largely unresolved until the 1990s. In the early 1990s, studies showed that this system regulates molecules critical for cellular function such as cyclins or p53 [[5, 6]]. Thus, I joined the ubiquitin field with good timing. IRP2 regulates cellular iron metabolism by interacting with mRNAs that encode proteins involved in iron metabolism under iron-depleted conditions. During the characterization of the iron-mediated regulation of IRP2, I found that the abundance of IRP2 is regulated by the iron-dependent degradation of the protein, which depends on cellular iron availability. I then used proteasome inhibitors [] to show that IRP2 is degraded by the proteasome [].
In most cases, ubiquitin modification is mediated by the conjugation of ubiquitin chains, which are polymers of ubiquitin. This feature makes ubiquitination distinct from other modifications. Ubiquitination is catalyzed by the repetitive function of three enzymes, namely E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). Ubiquitin is first conjugated to the ε-amino group of a lysine (K) residue in a protein, which is recognized by the E3s, leading to the successive conjugation of ubiquitin onto the ubiquitin moiety conjugated to the protein to generate ubiquitin chains (Fig. 1A). []. Among the enzymes of the ubiquitin conjugation system, E3 ubiquitin ligases, of which approximately 600 have been identified in humans, are considered the most important enzymes because they recognize substrate proteins in a timely and selective manner for ubiquitination.
In 1996, I left NICHD and joined the laboratory of Dr Nagahiro Minato in the Department of Immunology and Cell Biology of Kyoto University because I was still interested in immunology. Dr Minato was kind enough to allow me to continue ‘the IRP2 project’ during my stay at Kyoto. In his laboratory, I focused on identifying the E3 specific for IRP2 under conditions of iron repletion and found heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1) as a candidate E3 []. At the same time, we showed that pVHL, a tumor suppressor product of clear cell renal carcinomas, functions as the substrate recognition subunit of the Cullin/RING ligase complex; this work was performed in collaboration with Dr Arnim Pause, who was a postdoctoral fellow at the laboratory of Dr Klausner, and Dr Joan Conaway []. These studies allowed me to secure an independent position at Osaka City University, where I initiated ubiquitin projects in 2001.
I would like to introduce the status of ubiquitin research when I started working on ubiquitin at Osaka City University. Ubiquitin is conjugated in the form of a ubiquitin chain in most cases, and ubiquitin chains are generated via the Lys residues of ubiquitin and those of target proteins. Ubiquitin has seven Lys residues, and Lys 48 (K48)-linked chains serve as the recognition signal for the 26S proteasome []. The roles of other ubiquitin chains were discovered after 1995. Early studies showed that ubiquitin chains generated via Lys 63 (K63) of ubiquitin are involved in DNA damage responses in S. cerevisiae []. Later studies showed that K63 chains are involved in other signaling functions that do not affect the fate of proteins in mammals []. Mass spectrometric analyses revealed that ubiquitin is modified at all seven Lys residues to generate chains in S. cerevisiae []. Approximately 90 deubiquitinating enzymes have been identified in humans, supporting that ubiquitination is a reversible post-translational modification []. It started revealing that ubiquitin conjugation system plays diverse roles in physiology and medicine, in addition to its role in protein degradation when I started working on ubiquitin at Osaka City University.
Discovery of the linear ubiquitin chain and the LUBAC ligase complex
My work in Osaka City University involved investigating the HOIL-1 ubiquitin ligase. Immunoblotting using an antibody that recognizes the C terminus of HOIL-1 detected two discrete signals, which led us to search for isoforms of HOIL-1 []. A database search identified a longer isoform, HOIL-1L. Although HOIL-1L is a major form of HOIL-1, HOIL-1L and HOIL-1 did not account for the slower migrating signal observed by immunoblotting, suggesting that one of the HOIL-1 forms is modified post-translationally. Both isoforms of HOIL-1 have an RING-in between RING (IBR)-RING domain at the C terminus, which is the catalytic center of E3s. Because RING-IBR-RING (RBR) ligases function as monomers or homo-multimers, we suspected that a form of HOIL-1 may be modified in a large molecular weight complex. To test this, we performed gel filtration analyses, which showed that HOIL-1L, but not HOIL-1, forms a high molecular weight complex of approximately 600 kDa. The complex was identified as a hetero-multimer complex because overexpressed HOIL-1L formed a complex smaller than 600 kDa []. The slower migrating signal detected by immunoblotting was observed exclusively in the high molecular weight complex and appeared to be a modified form of HOIL-1L.
We then attempted to identify proteins in the complex containing HOIL-1L by purification from HeLa cell lysates. We identified a HOIL-1L interactor that was designated as HOIL-1L-interaction protein (HOIP). Further analysis showed that HOIP is also an E3 ubiquitin ligase containing the RBR domain. Co-introduction of cDNAs encoding HOIL-1L and HOIP revealed that HOIL-1L and HOIP generate a 600 kDa high molecular weight complex that co-migrated with the endogenous complex containing HOIL-1L. This led us to hypothesize that HOIL-1L forms a high molecular weight complex with HOIP []. We later identified SHANK-associated RH domain interactor (SHARPIN) as another protein interacting with HOIL-1L and HOIP [], although we failed to identify the interactor during the purification of HOIL-1L interacting proteins possibly because SHARPIN co-electrophoresed with β-actin.
Initially, the physiological function of the HOIL-1L/HOIP complex remained elusive. As shown in Fig. 1B, HOIL-1L and HOIP have multiple domains in addition to the RBR domain. Among these, the ubiquitin-associated (UBA) and Npl4 zinc finger (NZF) domains are ubiquitin-binding domains. Therefore, the HOIL-1L/HOIP complex has both E2-binding and ubiquitin-binding sites, which gave me a clue to dissect its molecular function of the complex.
Elucidating the molecular mechanism underlying the conjugation of ubiquitin chains to substrates remains a challenge in the ubiquitin field. The current theory is that the repetitive function of the three enzymes, E1, E2, and E3, mediates the conjugation of ubiquitin chains to substrates recognized by E3s. In the first reaction, ubiquitin is conjugated to a Lys residue in the substrate, which is followed by conjugation of ubiquitin to a Lys residue of another ubiquitin moiety. Therefore, two different reactions can be catalyzed by the three enzymes. Because E3s recognize substrates, the spatial relation between enzymes (E3s) and the sites of reaction (interubiquitin linkage) changes during the chain elongation step (Fig. 1A). This phenomenon contradicts the principles of enzymology because enzymes facilitate reactions by recognizing a substrate through its catalytic site to reduce the energy of reaction intermediates.
Because the HOIL-1L/HOIP complex contains the RBR E3 and ubiquitin-binding domains, we speculated that this ligase complex recognizes ubiquitin and mediates the conjugation of ubiquitin. This would imply that the reaction site does not change during the chain elongation reaction because the complex preferably recognizes distal ubiquitin moieties of chains. To test this hypothesis, we designed in vitro ubiquitination assays using artificial substrates, such as GST and GFP fused to ubiquitin at the N terminus (Ub-GST and Ub-GFP). The results showed that the HOIL-1L/HOIP ligase complex successively conjugated ubiquitin to ubiquitin-fused artificial substrates but not to nonfused ones to generate ubiquitin chains [].
The next obvious question was the type of ubiquitin chain generated by the complex because all seven Lys residues in ubiquitin are capable of generating linkages, as previously determined using mass spectrometry []. To test this, we performed in vitro ubiquitination assays using ubiquitin mutants because ubiquitin is a small protein that can be expressed and purified using an E. coli expression system. Ubiquitin mutants of one of seven Lys residues and a Lys-less (K0) mutant were prepared. The K0 ubiquitin mutant should be unable to generate any ubiquitin chains because ubiquitin chains are generated via Lys residues. However, we found that the HOIL-1L/HOIP ligase complex was able to generate ubiquitin chains from K0 ubiquitin. Mass spectrometry analysis of the in vitro-generated chains showed that the HOIL-1L/HOIP ligase complex catalyzed the conjugation of the C-terminal carboxyl group of a donor ubiquitin and the α-amino group of the N terminus methionine of an acceptor ubiquitin. In vitro ubiquitination assays using methylated ubiquitin, in which the N-terminal α-amino group and the ε-amino groups of Lys are methylated, did not generate ubiquitin chains, confirming that the HOIL-1L/HOIP complex generated ubiquitin chains via the N terminus of ubiquitin. These chains were termed linear ubiquitin chains (or M1 chains), and the ubiquitin ligase complex was named linear ubiquitin chain assembly complex (LUBAC) [].
LUBAC contains two RBR E3 catalytic centers, namely the RBRs of HOIP and HOIL-1L. Although the precise catalytic mechanism of RBR ligases was undefined when we identified LUBAC, the RING domain of RING family ligases is considered the binding domain for E2s. Substitution of critical Cys residues of the RING domain in HOIP or HOIL-1L indicated that the RBR of HOIP is the catalytic site for linear ubiquitination. To identify the E2s that function in conjunction with LUBAC to generate linear ubiquitin chains, we performed in vitro ubiquitination assays. The results showed that several E2s, including UBE2D1-3 (UbcH5a-c), UBE2L3 (UbcH7), and UBE2K (E2-25K), could generate linear chains. The fact that UBE2K could generate linear chains with LUBAC was an unusual finding because UBE2K is a unique E2 that generates K48 chains in the absence of an E3 []. LUBAC exclusively generated linear chains even in the presence of UBE2K, suggesting that LUBAC, but not E2s, determines the type of chain formed []. To the best of our knowledge, this was the first report showing that E3s determine the type of chain generated. More importantly, ubiquitin chains have been believed to be generated via one of the seven Lys residues of ubiquitin at that time. Indeed, no linear chains are found in S. cerevisiae. This suggested that we identified a novel ubiquitin ligase complex that specifically generates unique M1-linked linear ubiquitin chains using biochemical, not genetic, analysis of the HOIL-1L ligase. So far, the LUBAC ligase complex, after identification of SHARPIN as the third subunit of the ligase complex as discussed later [], is the only E3 that generates linear chains.
Physiological functions of LUBAC-mediated linear ubiquitination
Identification of the linear ubiquitin chain and the LUBAC ligase led us to investigate the physiological functions of LUBAC-mediated linear ubiquitination, which was a challenging task. The X protein of hepatitis B virus (HBx), a HOIL-1-binding protein [], was used as bait in 2-hybrid screening, which led to the identification of Murr1 (COMMD1). Murr1 is a causative gene product for copper toxicosis in dogs. My associate, Dr Fuminori Tokunaga, notified me of this protein as an HBx-binding protein since we also worked on metal ions. But, I neglected it at first because it seemed unrelated to LUBAC function. A couple of months later, however, a paper was published showing the possible involvement of Murr1 in nuclear factor-κB (NF-κB) signaling [], which prompted me to examine the involvement of LUBAC in NF-κB activation. The ubiquitin system is involved in the activation of NF-κB, which plays crucial roles in various processes including inflammation and oncogenesis []. A laboratory near our group that was working on cytokine-induced transcriptional activation in immune cells provided several luciferase reporter constructs including one for NF-κB, which we used to determine whether HOIL-1L could activate NF-κB []. The results showed that HOIL-1L alone did not activate NF-κB. However, because of the recent identification of the LUBAC ligase complex, we examined its possible involvement, and the results showed that co-introduction of HOIL-1L and HOIP activated NF-κB [].
In quiescent cells, NF-κB is retained in the cytoplasm through binding to the inhibitor of κB (IκB). Upon exposure to stimuli including TNF-α or IL-1β, the IκB kinase complex (IKK), which is composed of IKK1, IKK2, and NF-κB essential modifier (NEMO), is activated by phosphorylation of IKK2, leading to phosphorylation and degradation of IκB and the subsequent activation and nuclear translocation of NF-κB []. We showed that LUBAC catalyzes the conjugation of linear ubiquitin chains onto NEMO in the IKK complex, which activates IKK and induces NF-κB nuclear translocation []. Work by the laboratories of Drs Hao Wu and Ivan Dikic independently showed that specific recognition of linear diubiquitin by the UBD in ABIN proteins and the NEMO (UBAN) domain of NEMO functions in NF-κB activation [[24, 25]]. Our group elucidated the molecular mechanism underlying LUBAC-mediated NF-κB activation as follows: The linear ubiquitin chains conjugated onto NEMO by LUBAC are recognized by the UBAN domain of NEMO in another IKK complex, which leads to the activation of IKK2 via dimerization of the kinase in different IKK complexes, which leads to IKK2 autophosphorylation and subsequent activation of NF-κB [[26-28]]. Apoptosis was increased in cells with decreased LUBAC activity, consistent with the involvement of NF-κB in cell death suppression []. LUBAC-mediated linear ubiquitination of the components of the activated TNF receptor complex attenuated the production of death-inducible complex II, which comprises receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, FAS-associated death domain protein (FADD), cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP), and caspase-8, thereby suppressing death receptor-induced programmed cell death, including apoptosis and necroptosis (Fig. 2) [].
Involvement of linear ubiquitin chains in human diseases
SHARPIN, which was identified as the third component of LUBAC, is involved in immunological diseases [[17, 30, 31]]. We first identified SHARPIN as a HOIL-1L-binding protein in a yeast 2-hybrid screening. We then hypothesized that SHARPIN is the third component of LUBAC because of the similarity between the ubiquitin-like (UBL) domain of SHARPIN and the HOIL-1L UBL, which is the binding site for HOIP, and it turned out to be the case. In 2007, Sharpin was identified as a causative gene of chronic proliferative dermatitis in mice (cpdm) [], which was described as a spontaneous mutation in mice exhibiting chronic skin inflammation in 1993 []. In addition to dermatitis, cpdm is associated with chronic inflammation of multiple organs and immunodeficiency []. Deletion of SHARPIN drastically reduces the amount of the LUBAC complex composed of HOIL-1L and HOIP, suggesting that reduction in the cellular linear ubiquitination activity of LUBAC underlies the pathogenesis of cpdm []. Decreased LUBAC function is also associated with human diseases; for example, mutations in genes encoding LUBAC subunits such as Hoil-1l (Rbck1) and Hoip (Rnf31) cause human immunodeficiency and autoinflammation [[34, 35]]. The deubiquitinating enzyme (DUB) that cleaves linear ubiquitin chains, OTU deubiquitinase with linear linkage specificity (OTULIN) [[36, 37]], is associated with autoinflammatory diseases also by attenuating LUBAC functions []. OTULIN interacts with LUBAC via the peptide:N-glycanase and UBA or UBX-containing proteins (PUB) domain of HOIP [].
LUBAC dysregulation is also involved in oncogenesis. Rare germline polymorphisms of Hoip (Rnf31) are highly enriched in patients with the B-cell-like type of diffuse large B-cell lymphoma (ABC-DLBCL). Because constitutive activation of NF-κB is the hallmark of ABC-DLBCL [], we further examined the effect of HOIP polymorphisms and showed that they increased LUBAC activity by promoting the interaction between HOIL-1L and HOIP []. We further dissected the role of LUBAC-mediated linear ubiquitination in B lymphoma in mice overexpressing HOIP in B cells. Increased LUBAC activity facilitated B lymphomagenesis by preventing cell death induced by DNA damage and by increasing NF-κB signaling []. Furthermore, a natural compound that specifically inhibits LUBAC suppresses tumor growth in a mouse transplantation model []. LUBAC is also involved in resistance to a widely used anticancer drug, cis-platinum [].
Gliotoxin, a crucial virulence factor of the opportunistic pathogen Aspergillus, inhibits LUBAC []. In addition, certain pathogens, including Shigella and Legionella, inhibit LUBAC to maintain infectability [[44, 45]]. Taken together, these studies indicate that LUBAC-mediated linear ubiquitination is involved in immune-related disorders, tumor development, and infection.
Regulatory mechanism of LUBAC-mediated linear ubiquitination
After the identification of the LUBAC ubiquitin ligase complex, we focused on structural and functional analyses of LUBAC. Because loss of SHARPIN drastically destabilizes LUBAC [], we explored the mechanism underlying the formation of the stable trimeric LUBAC. The interaction between the UBA domain of HOIP and the UBL domains of HOIL-1L and SHARPIN plays a crucial role in LUBAC formation []. However, biochemical analyses indicate that regions of N-terminal to the UBL of both HOIL-1L and SHARPIN are also critical for the stability of trimeric LUBAC. In collaboration with Dr Masahiro Shirakawa, we used X-ray structural analyses to show that LUBAC-tethering motifs (LTMs) located N-terminally to the UBL of HOIL-1L and SHARPIN heterodimerize and fold into a single globular domain []. Since LTM fragments from two different proteins are folded into a single globular domain, LTM-mediated interaction plays a critical role in stabilizing trimeric LUBAC because of its resistance to dissociation. Our original identification of SHARPIN as a binding protein of HOIL-1L in yeast 2-hybrid analyses was inconsistent with the fact that interaction between the two proteins was not observed in co-immunoprecipitation analyses at first []. This may be explained by the increase in LTM-mediated dimerization of HOIL-1L and SHARPIN in the presence of HOIP []. The marked reduction in LUBAC caused by chemical inhibition of LTM-mediated HOIL-1L/SHARPIN dimerization supports the important role of LTM-mediated dimerization in the stability of LUBAC [].
A substantial amount of HOIL-1L in LUBAC is modified post-translationally [], and modification of HOIL-1L disappears when HOIL-1L E3 activity is lost. We then dissected the roles of HOIL-1L E3 played in LUBAC function and found that loss of HOIL-1L E3 function promotes LUBAC-mediated NF-κB activation and suppression of programmed cell death []. Based on these findings, we explored the molecular mechanism underlying the suppression of LUBAC function by the HOIL-1L E3. The results can be summarized as follows []: HOIL-1L monoubiquitinates HOIL-1L as well as HOIP and SHARPIN, and the monoubiquitinated LUBAC subunits are recognized by HOIP to generate linear chains. Monoubiquitin conjugated onto LUBAC subunits by HOIL-1L renders the subunits to the suitable substrate for HOIP because HOIP specifically recognizes acceptor ubiquitin moieties with a linear ubiquitin chain-determining (LDD) domain and conjugates the donor ubiquitin onto the N terminus of the acceptor ubiquitin to form linear ubiquitin chains [[48, 49]]. Conjugation of linear chains onto LUBAC (autolinear ubiquitination) by the HOIP E3 suppresses LUBAC function, as demonstrated by the effect of OTULIN on increasing LUBAC activity by trimming linear chains conjugated onto LUBAC subunits []. Thus, our analyses of LUBAC regulation revealed a novel regulatory mechanism of E3s. The accessory E3 center of LUBAC (HOIL-1L RBR) monoubiquitinates LUBAC subunits, which makes the subunits preferred substrates for autolinear ubiquitination by the main E3 of LUBAC (HOIP RBR), thereby attenuate LUBAC function, while OTULIN, DUB specific for linear chains, maintains LUBAC function by trimming the chains. Thus, loss of HOIL-1L E3 activity promotes LUBAC function by suppressing the modification of LUBAC by linear ubiquitination. A HOIL-1L mutant lacking E3 activity improves cpdm dermatitis, which is caused by reduced amounts of the LUBAC complex composed of HOIL-1L and HOIP, and protects cells against Salmonella infection [].
I started my career as a physician specialized in rheumatology. However, several critical events led me to the discovery of linear ubiquitination. The first event was joining the laboratory of Dr Klausner as a postdoctoral fellow, which resulted in my change in major from immunology to iron metabolism. The second event was my work investigating the intracellular protease calpain as an undergraduate. When I was in the second grade, I received a phone call from a professor at Kyoto University, a good friend of my father, who said, ‘Is one of my friends interested in working in his laboratory to study calpain?’ I could not find any good candidates, and so, I offered to work on calpain in his laboratory during the summer recess of the third grade. My experience working on proteases shifted my interest from iron metabolism to the ubiquitin–proteolytic system. The third and possibly most important turning point was attending the FASEB meeting on ubiquitin in 1995. In that meeting, I met Dr Aaron Ciechanover, a good friend of Dr Klausner. Dr Ciechanover, together with Dr Avram Hershko (who are both Nobel laureates in chemistry), discovered the ubiquitin–proteasome system when searching for the answer to a simple question: ‘Why is energy required for proteolysis in cells?’ I was deeply impressed by their research attitude, which led me to address a purely biochemical issue, namely ‘How are ubiquitin chains generated via the repetitive functions of three enzymes?’ This resulted in my decision to pursue a career as a researcher. Several lucky events led me to discover the linear ubiquitin chain and LUBAC, and I followed this by investigating the molecular function and regulation of LUBAC. The role of LUBAC-mediated linear ubiquitination in immune signaling and oncogenesis is currently well-established. Both impairment and overactivation of the linear ubiquitin chain assembly complex cause diseases in humans, and numerous pathogens infect organisms by targeting LUBAC-mediated linear ubiquitination. Thus, LUBAC is a suitable target for infections, immunological diseases, and cancer. Our studies provide unique strategies to inhibit as well as promote LUBAC-mediated linear ubiquitination, and I expect that the discovery and elucidation of LUBAC-mediated linear ubiquitination will further contribute to human welfare.
The author is grateful to members of the Iwai laboratory for valuable discussions and insightful comments. Work in the Iwai laboratory is partly supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant Numbers: 24112002, JP17H06174, and JP18H05499) and the Takeda Foundation (to KI).
Conflict of interest
The authors declare no conflict of interest.
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