**5. Ubiquitination in Immune-Related Diseases**

Ubiquitination is one of the most essential post-translational modifications, playing an important role in both innate and adaptive immunity. Notably, E3 ligases have a major role in leukocytes activation, di fferentiation, and development [155]. Besides, DUBs, particularly USPs, are known as an important modulator for T cell function [156]. In accordance, recent studies demonstrated that defective UPS, including aberrant E3 ligases, DUBs, or proteasomes tend to the impairment of immune regulation and thereby cause the development of multiple inflammatory or autoimmune diseases [157].

Casitas B-lineage lymphoma (Cbl) proteins, consisting of Cbl-b, Cbl-3, and c-Cbl, are a member of the RING finger-containing E3-ligase superfamily [158]. Due to their RING finger domain, they serve as an E3 ligase to various substrates, forming stable interaction with E2-conjugating enzymes and subsequently promoting ubiquitin transfer from the E2 enzyme to their substrate. In previous studies, Cbls was revealed as a major modulator in immune regulation during the early development of hematopoietic precursor cells into the e ffector immune cells [159]. *Cb*−/− transgenic mice models show that the amount of CD4+ SP thymocytes are significantly elevated by ubiquitination of the CD3 ζ chain of the T-cell receptor (TCR) complex [160]. In B cell development, *Cbl*-deficient B cells show an improvement of BCR (B-cell antigen receptor) signaling and promote several downstream processes, such as Ca+<sup>2</sup> influx, Syk, and CD79A. Therefore, Cbl is known as a negative regulator of B and T cell development via ubiquitin-dependent degradation [161]. In addition, Cbls are known to regulate the activation of macrophages regarding immune disorders, particularly in cancer and obesity. For instance, *Cbl*−/− mice show enhanced tumor growth in colorectal cancer, and the recovery of Cbl expression increases the tumor cell phagocytosis of macrophages through the ubiquitination of surface proteins [162]. In obesity, Cbls act as negative regulators for macrophage activation by suppressing migration signals to adipose tissue. Therefore, insulin resistance and obesity are induced in *Cbl-*/*-* mice. Stimulated macrophage accumulation in adipose tissue results in the secretion of inflammatory cytokines in this model [163,164]. Although there are several studies about Cbls in macrophage regulation, molecular mechanisms underlying inflammatory diseases are still poorly understood.

Cbl-b works with other E3 ligases, which are known as Itch, to prevent the over-reactivity of T-cell-dependent peripheral tolerance. Itch is a monomeric protein, namely HECT (homologous to the E6AP carboxyl terminus), presenting intrinsic catalytic activity. Its main role in immunity is regulating immune cell development and function by mediating protein ubiquitination [155]. Recent studies show that Itch and Cbl-b collaboration leads to proteolysis, independent of ubiquitination of the TCRζ chain. Thus, the signal transduction of the TCR complex is suppressed by preventing ZAP70 and TCRζ chain binding [165]. The cooperative functions of di fferent E3 ligases support the regulation of immune cell homeostasis by ubiquitination [166].

Von Hippel-Lindau (VHL) is an E3-ligase complex, consisting of elongin B, elongin C, cullin 2, and ring box protein 1 (RBP1). These ligases are mainly regulated by Hypoxia-inducible factor-1 α (HIF-1 α), which functions as a critical transcription factor in immune regulation under hypoxic conditions. Under normal conditions, the VHL ligase attaches ubiquitin tags on HIF-1 α and prevents target gene activation [167]. In fact, HIF-1 α is responsible for the immunosuppressive function of regulatory T cells (Treg) [168]. In VHL deficiency, HIF-1 α promotes the elevation of interferon-γ (IFN-γ) production in Treg cells, which causes the emergence of the Th1-like inflammatory phenotype [169], indicating the critical role of the VHL ligase–HIF-1 α axis in T cell immunity and di fferentiation [156]. Low oxygen tension also a ffects innate immunity via HIF-1 α-dependent signaling. An impaired maturation of alveolar macrophages and suppression of neutrophil apoptosis were demonstrated in hypoxia or VHL-deficient conditions [170]. These findings support that the VHL/HIF-1 α pathway is responsible not only for T and B cell di fferentiation but also myeloid cell function during an innate immune response [171]. Moreover, hypoxia and VHL ligases are commonly associated with renal carcinoma. It has been shown that VHL expression is negatively correlated with tumor malignancy through promoting immune responses [172] and in patients, mutated VHL ligase proteins were found to show more natural killer cell toxicity [173]. Taken together, VHL E3 ligases appears to be a remarkable protein for immune regulation and immune-related disease prevention.

Since ubiquitination is a reversible process, deubiquitinases play a crucial role in maintaining an effective immune response, particularly for constituting adaptive immunity [156]. USPs are the largest family of DUBs including at least 50 members; however, the most important USPs in immune regulation are USP4, USP8, USP9X, USP12, and USP19 [75,174], which are mainly involved in T cell homeostasis as positive or negative regulators. Specifically, USP4 is responsible for promoting Th17 cell di fferentiation and function. The most established transcription factor RORγ<sup>t</sup> (RAR-related orphan receptor gamma) stabilization for Th17 cell di fferentiation is mediated by highly expressing USP4. Hence, USP4 was suggested as a potential target for inhibiting Th17-mediated autoimmune disorders, such as rheumatic heart disease [175]. USP8 is another important member of DUBs, which are critical for T cell maturation. The knockdown of T cell-specific USP8 showed that the maturation and proliferation of thymocytes were impaired by a Foxo1–IL-7R α-dependent mechanism. USP8 mutant mice developed lethal colitis because of impaired T cell homeostasis [176]. Moreover, somatic mutations in the human USP8 gene were suggested to suppress the immune system, which causes the development of Cushing's Disease. These mutations are associated with the 14-3-3 binding motif (RSYSS) of USP8, which is important for its phosphorylation; thus, mutations in RSYSS motif lead to impairing its DUB activity. In patients with Cushing's disease, it was observed that adrenocorticotropic hormone (ACTH) is produced at a higher level because of USP8 mutations [177]. Similar to USP8, USP9X acts as a positive mediator for TCR signaling and T cell tolerance. The loss of USP9X leads to the decrease in T cell proliferation and T cell di fferentiation into helper T cells, hence a ffecting cytokine production. USP9X deficiency in T cells attenuates TCR signaling and promotes the activation of the NF-κB pathway because of modulating upstream signaling proteins of the NF-κB [178]. USP12 deubiquitinates some of the TCR adaptor proteins, such as LAT (Linker for Activation of T cells) and Trat1, stabilizing the TCR:CD3 complex on the cell surface and consequently preventing the lysosomal degradation of LAT and Trat1 [179]. On the other side, USP19 has a di fferent role in immune regulation compared to other USPs discussed above, since it is related to an innate immune response with its Toll-Like Receptor (TLR)-dependent activity. In a very recent study, Usp19−/− mice treated with poly(I:C) or LPS showed the elevation of pro-inflammatory cytokines and type 1 interferons secretion, indicating that USP19 negatively regulates TLR3/4-mediated signaling by impairing the accession of the essential adaptor protein to TLR3/4 [180].

Cylindromatosis (CYLD) was first discovered as a tumor-suppressor protein [181]. However, it has been shown that CYLD belongs to the deubiquitinating enzyme family, and it removes the Lys63-linked polyubiquitin chain from signaling proteins located upstream of NF-κB, including TNF receptor-associated factor 2 (TRAF2), TRAF6, and NEMO [182]. It is well known that CYLD is a critical factor for T cell development, activation, and function. In fact, the essential role of CYLD in T cell development was identified with threshold activation for the positive selection of T cells via NEMO-dependent NF-κB signaling. In an in vivo study, *Cyld*-deficient mice developed a chronic inflammatory digestive disease, colitis [183]. The underlying mechanism is that CYLD targets TGF-β-activated kinase 1 (TAK1) and inhibits its ubiquitination and autoactivation. This mechanism

also activates downstream kinases of TAK1 such as c-Jun N-terminal kinase (JNK) and IκB kinase β (IKKβ) due to the autoactivation of TAK1 [184,185]. In line with this information, the function of CYLD in T cell regulation is considered as a negative feedback mechanism by inhibiting activation of the TAK1 axis of TCR signaling. In transgenic mouse models of CYLD, changes in immune cell function, abnormal hepatocytes, hair, and dental defects were observed [185–187]. Furthermore, CYLD polymorphisms were specifically associated with inflammatory bowel disease (IBD) that comprised 2320 patients with Crohn's disease [188].

UPS has been shown as the main modulator for the antigen presentation in immune regulation. More specifically, oligopeptides are hydrolyzed by the proteasome after binding PA28, an activator for hydrolysis, to the ends of 20S proteasome. The process is controlled by a proteolytic cascade including aminopeptidases that are responsible for peptide production for the presentation of MHC-I (Major Histocompatibility Complex-I) [189]. Notably, the MARCH (Membrane-associated RING-CH-type finger) E3 ligase is involved in the reduction of MHC-I surface expression by mediating the polyubiquitination of MHC-I. Therefore, MARCH-mediated MHC presentation is crucial for UPS function in immune regulation [190]. Interestingly, autoantibodies, reacting as self-antigens, were detected in 20S proteasome subunits in several autoimmune diseases, such as systemic lupus erythematosus, primary Sjögren's syndrome, and myositis. In vitro studies showed that autoantibodies can inhibit proteasome activation by targeting the PA28 binding. On the other hand, proteasome levels in circulation are suggested as an important biomarker for disease progression and potential drug targets, because of their increased levels observed in autoimmune myositis, systemic lupus erythematosus, primary Sjögren's syndrome, and rheumatoid arthritis [191,192].
