Immunological response, which is a body’s critical defense mechanism against pathogens and foreign substances, is tightly controlled at multiple levels including reversible modifications of various signaling molecules. Ubiquitination is an important post-translational modification that covalently attaches ubiquitin chains to target molecular proteins and either marks substrate proteins for proteasomal degradation or allows them to function as a scaffold to recruit other proteins and mediate downstream signaling (1–3). Linear ubiquitin chains are a unique type of ubiquitin chain, in which the ubiquitin molecules are linked together in a linear fashion through their N-terminal methionine and C-terminal glycine residues, and are generated by a multi-subunit E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC), which compromises three subunits: HOIL-1L (heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1 like, also known as RBCK1), HOIP (HOIL-1L-interacting protein, also known as RNF31), and SHARPIN (SHANK-associated RH domain-interacting protein) (4, 5). Numerous publications reported functions of linear ubiquitin chains including regulating immune responses and inflammation, via NF-κB signaling in response to various stimuli, and preventing TNF-induced cell death (6–13). That disruption of LUBAC components leads to immunodeficient disorders and autoinflammatory disorders in humans (14), and embryonic lethality or inflammatory phenotype in mice (11, 12, 15), highlights the crucial role of LUBAC-mediated Met1 signaling in maintaining normal mammalian physiology. Ubiquitination is reversed by the deubiquitinating enzyme (DUB) via cleaving the ubiquitin molecules from their substrate (2). To date, almost 100 DUBs are identified in humans (2), but mainly A20, cylindromatosis (CYLD), and OTULIN have been characterized as key DUBs involved in the negative regulation of NF-κB activation and cell death (16). OTULIN, also known as Fam105B or Gumby, was recently identified in human and mice (17, 18), with exclusive specificity to hydrolyze Met1-poly Ub chains (4, 17–19). Herein, we briefly review the genetic and clinical features of OTULIN deficiency, focusing on immunological phenotypes. Finally, we summarize the state-of-the-art management options, shedding light on the current main research gaps.

The 352-amino acid protein OTULIN was initially discovered by Keusekotten et al. through a bioinformatics approach (17, 18, 20). This important deubiquitinating enzyme comprises a catalytic ovarian tumor (OTU) protease domain, an N-terminal interaction motif (PIM) involving peptide:N-glycanase (PNGase)/UBA-containing or UBX-containing proteins (PUB), and a C-terminal PSD95-Dlg1-ZO-1(PDZ) domain-binding motif (17, 18, 21, 22). OTU domain spans 80–345 amino acids, shares features of the papain-like family of cysteine peptidases, and employs a Cys129/His339/Asn341 catalytic triad (17, 20) ( Figure 1 ). A striking function of OTULIN is its exclusive hydrolyzation to Met1-linked poly-Ub chains, without affecting other linkage types of poly-Ub chain (17, 18). This feature function originates from a highly conserved Ub binding site located in the OTU domain via a mechanism known as substrate-associated catalysis (17). Upon binding to Met1-linked poly Ub chains, the S1’ binding site in OTULIN orientates the Met1 residue of the proximal Ub toward the catalytic center, while the inter-Ub Lys residues remain remote from the catalytic center (17). However, binding of Ub chains with different linkages, but structurally similar Lys63-linked chains, would rotate the proximal Ub for several degrees and restrict proper binding to the S1’ binding site (17, 20). Ub substrates have also been demonstrated to directly contribute to OTULIN catalytic activity (17). In the absence of Ub, OTULIN Cys129, His399, and Asn341 adopt an inhibited catalytic triad conformation, and this auto-inhibition is released upon binding to Met1-linked proximal Ub (17). In addition, OTULIN selectively interacts with the LUBAC HOIP PUB domain via the N-terminal PIM, which covers Asp54–Met55–Tyr56–Arg57–Ala58 residues (21, 22) and serves as the sole site of interaction between OTULIN and HOIP, whereas the OTU domain’s presence is unnecessary for binding to HOIP (21, 22). Mutations in the OTULIN catalytic Cys129 do not affect the association between OTULIN and HOIP (15, 21).

Pathogens, tissue damage, and stress provoke host immune response and activate the downstream inflammatory signaling pathway. While immune receptors, such as TNFR1, NOD-like receptors (NLRs), CD40, toll-like receptors (TLRs), and the interleukin-1 receptor (IL-1R) instigate the assembly of receptor signaling complexes (RSCs), LUBAC recruitment via a Lys63 ubiquitin-dependent approach is a pivotal step in the course of these complex processes (5, 23–26). LUBAC recruitment facilitates Met1-linked ubiquitination of TNFR1, receptor-associated proteins, or pre-existing Lys63 polyubiquitin chains, and initiates downstream MAPK and NF-κB signaling by orchestrating the recruitment of the TAK1 and IKK complexes (24, 25, 27, 28). Numerous studies have demonstrated OTULIN’s role in counteracting LUBAC-mediated Met1 ubiquitination and subsequent NF-κB activation (17, 18, 29–33). However, deficiencies in OTULIN, overexpression of catalytic inactive OTULIN mutants, and OTULIN knockdown result in a remarkable increase in cellular Met1 Ub levels, leading to the accumulation of Met1-linked Ub on several pivotal proteins within inflammatory pathways, including TNFR1, receptor-interacting serine/threonine-protein kinase-1 (RIPK1), receptor-interacting serine/threonine-protein kinase-2 (RIPK2), NF-κB essential modulator (NEMO), and LUBAC (17, 23, 31–35). By contrast, overexpression of OTULIN leads to a reduction in TNFα-induced NF-κB activation and gene transcription (17). It is worth noting that unlike CYLD and A20, which exert their influence on NF-κB signaling through negative feedback mechanisms, OTULIN is not subject to transcriptional regulation by NF-κB (17, 18, 32, 36, 37).

The OTULIN gene is located on chromosome 5p15.2. To date, approximately 99 pathogenic or likely pathogenic germline variants are identified, affecting nearly 50 individuals (29–31, 38–40). Patients with variants in OTULIN gene and size of variants larger than 50 bps mainly present 5p- syndrome and are responsible for 72% of cases. Single genetic variants in OTULIN only account for 20%–30% of cases. Among these cases with single genetic mutation in OTULIN, missense mutations in OTULIN clustered within the OTU region underlie roughly 62.5% of cases, whereas null mutations (frameshift, nonsense, and splice site), located prior to or within the OTU region, represent approximately 37.5% of mutations (29–31, 39, 40). The impact of mutations in the OTULIN gene is quite variable, resulting in a range of clinical phenotypes. Loss-of-function mutations in the OTULIN gene often led to reduced levels of the OTULIN protein, which, in turn, contributes to the observed variability in clinical presentation. However, some mutations, such as OTULIN (L272P), are associated with only minimal degradation of the OTULIN protein (30, 31). Others, like OTULIN (G281R), result in almost complete loss of function (29). These findings underscore the complex and diverse effects of OTULIN gene mutations on protein expression and function, highlighting the need for further research to fully understanding the underlying mechanisms and potential therapeutic implication.

Given the broad spectrum of clinical manifestations, the diagnosis of OTULIN deficiency can be challenging. A strong clinical suspicion must arise when clinical features, such as neonatal onset of recurrent fever, erythematous rash with painful nodules, and painful joints, are presented. Because of the auto-inflammatory nature of ORAS, anti-inflammatory drugs currently represent an effective treatment. However, treatment of affected patients with prednisolone (general cortico-steroidal immunosuppressant), azathioprine, methotrexate (anti-proliferative immunosuppressive drugs), and Anakinra (recombinant IL-1R-antatonist) had little or no effect on clinical symptoms (30, 31). By contrast, TNF-blocking agents infliximab or etanercept drastically reduced the symptoms, and serum levels of CRP as well as WBC and neutrophil counts in blood returned to normal ranges, suggesting a TNF-mediated pathogenesis in the patients (30, 31). Hematopoietic stem cell transplantation (HSCT) has been treated in one patient who developed severe inflammatory symptoms after birth, including fever and panniculitis (29). At 7 months of age, this patient had fever, diarrhea, panniculitis, cachexia, and cataracts. She underwent an HSCT at 17 months of age, and her symptoms resolved for 9 months, at which point she relapsed with fever and panniculitis. It was found that she had a decrease in bone marrow chimerism and regrowth of native hematopoietic cells. At 29 months of age, she was treated with Etanercept and had resolution of symptoms (29).

Further research needs to explore effective treatments other than anti-inflammatory drugs. Gene editing technology, such as the CRISPR/Cas9 system, represents one of the most curative approaches, in the wake of a successful outcome in the treatment of other hereditary diseases. In addition, the use of homology-direct repair and somatic genetic rescue as a correction mechanism may be attempted.

Germline mutations of OTULIN cause various complex clinical phenotypes, including autoinflammatory disorders and immunodeficient disorders. Despite the emergent findings, the exact pathogenetic details underlying these genetic variants are elusive. Uncovering these subtle molecular mechanisms will help develop more efficient and targeted treatments for patients.

CL: Funding acquisition, Supervision, Visualization, Writing – review & editing, Writing – original draft. BD: Investigation, Writing – original draft, Data curation. WP: Writing – original draft. GJ: Investigation, Data curation, Writing – original draft.