Tripartite motif family proteins in inflammatory bowel disease: Mechanisms and potential for interventions

Abstract Inflammatory bowel disease (IBD) is a chronic recurrent gastrointestinal inflammatory disease that poses a heavy burden to the global healthcare system. However, the current paucity of mechanistic understanding of IBD pathogenesis hampers the development of aetiology‐directed therapies. Novel therapeutic options based on IBD pathogenesis are urgently needed for attaining better long‐term prognosis for IBD patients. The tripartite motif (TRIM) family is a large protein family including more than 70 structurally conservative members, typically characterized by their RBCC structure, which primarily function as E3 ubiquitin ligases in post‐translational modification. They have emerged as regulators of a broad range of cellular mechanisms, including proliferation, differentiation, transcription and immune regulation. TRIM family proteins are involved in multiple diseases, such as viral infection, cancer and autoimmune disorders, including inflammatory bowel disease. This review provides a comprehensive perspective on TRIM proteins' involvement in the pathophysiology and progression of IBD, in particular, on intestinal mucosal barriers, gene susceptibility and opportunistic infections, thus providing novel therapeutic targets for this complicated disease. However, the exact mechanisms of TRIM proteins in IBD pathogenesis and IBD‐related carcinogenesis are still unknown, and more studies are warranted to explore potential therapeutic targets of TRIM proteins in IBD.


| INTRODUCTION
Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disease of the gastrointestinal tract that can be subdivided into Crohn's disease (CD) and ulcerative colitis (UC). 1 CD is distinguished by transmural inflammation and skip lesions distributed throughout the gastrointestinal tract, usually complicated by perianal lesions. UC associated inflammation is generally confined to rectum and colon and afflicts only the superficial mucosa, which usually demonstrates mucopurulent bloody stool. 2,3 The prevalence of IBD is as high as 0.5% in western countries, which has brought huge expenditure and heavy burdens to the healthcare system. 4 In 2020, the annual direct expenditure of every UC patient in Europe was estimated as €2000, and that of CD was €3500. 5 Moreover, in newly industrialized countries with lower IBD prevalence, such as China and India, the large populations and rising incidence rates bring remarkably heavy burdens to society. 6 At present, the mainstream therapy for IBD focuses on immunosuppression, in which biological agents have significantly improved prognosis. 7 However, despite these advances, over 40% IBD patients still require at least one surgery during the course of the disease; moreover, Rirong Chen and Yizhe Tie contributed equally to this work. non-responses to biologics are reported more often than not. 8,9 Therefore, new therapies based on a mechanistic understanding of IBD pathogenesis are urgently needed to develop more potent treatment regimens and reap long-term clinical benefits for IBD patients.
The pathogenesis of IBD remains unclear; it has been generally recognized to be the result of complex interactions between multiple factors, including genetic susceptibility, environmental determinants, intestinal flora and an imbalance of innate and adaptive immunity. 10 Nowadays, a number of studies have demonstrated that the intestinal barrier performs an essential role in the pathophysiology of IBD. 11,12 This barrier encompasses four parts, the mechanical, biological, chemical and immune barriers, and IBD are characterized by the impairment of multiple components among them. 13 The tripartite motif (TRIM) protein family contains more than 70 members characterized by the RBCC structure, in which one or two variable C-terminal domains show high structural diversity that determines the functional specificity of each protein. 14 TRIM proteins mainly function as E3 ubiquitin ligases, thus participating in the ubiquitination process and regulating many important post-translational protein modifications. 15 TRIM family proteins not only play essential roles in many biological processes, including proliferation, differentiation, transcription and apoptosis, along with the regulation of immune responses, but also participate in many diseases, including cancer, infectious diseases, neurodegeneration and developmental diseases. 16,17 Recently, TRIM family proteins, such as TRIM20 and TRIM27, were demonstrated to regulate intestinal barrier function and get involved in the pathophysiology of IBD. This review summarizes the structures and functions of TRIM proteins and discusses the mechanisms underlying their role in IBD pathophysiology to provide a novel approach for the exploration of potential therapeutic targets for IBD.

| Structure of TRIM family proteins
In general, TRIM family members share a highly conservative and typical structure of "RBCC", 18 which means that the order of TRIM proteins, from N-to C-terminus, is a really interesting new gene (RING) domain, one or two B-box domains and a coiled-coil (CC) domain. The RBCC domain is usually followed by more divergent C-terminal domains, which determine the specificity of each TRIM protein. 19 Moreover, the subcellular distributions of TRIM proteins show high variability, with their presence being reported in the cytoplasm, nucleus and plasma membrane. 20 The RBCC motif, which generally consists of three specific domains, is present in almost all members of the TRIM family. 18 The RING domain is the foremost structure consisting of 40-60 amino acid residues with the zinc-finger structure that binds two zinc ions. 17 Some investigations have found that they specifically bind to E2 ubiquitin-conjugating enzymes to exert E3 ubiquitin ligase activity, thus mediating the conjugation of specific proteins with one of the most widely recognized posttranslational modifiers, ubiquitin. 17 The B-box domain consists of 32-42 residues located behind the RING domain, which can combine with one or two zinc atoms as well. 17 Depending on the number of residues, it can be divided into B-Box 1 and B-Box 2. 21 23,24 Moreover, some researchers hypothesize that B-box enhance the recognition of target proteins. 25 The coiled-coil (CC) domain is the third characteristic structure of TRIM proteins, which is known to mediate the homologous or heterogeneous oligomerization of TRIM proteins and has a specific subcellular localization function. 26 Unlike the highly conserved RBCC sequences, the C-terminal domain behind the RBCC domain is highly variable; they have been implicated in substrate recognition and serve as a binding site for different targets. 27,28 So far, a total of ten types of C-terminal domains have been identified by structural analysis, and different combinations of them allow TRIM proteins to be categorized into 11 distinct subclasses (C-I to C-XI in Table 1), which may contain no or as many as three C-terminal domains. 36 In addition, a specific set of TRIM proteins which lack the RING domain are classified as the uncategorized group (Table 1). 27 The most prevalent C-terminal domain supposes to be SPRY, which exists in more than half of the TRIM family members and sometimes coexists with the PRY domain. 19 The SPRY domain mediates the recognition of and interaction with target proteins or RNA and regulates host-pathogen interactions and innate immune responses. 37,38 The C-terminal subgroup one signature (COS) domain is another frequently encountered C-terminal domain, which can bind to the microtubule cytoskeleton and participate in homodimerization or heterodimerization. 39 The fibronectin type 3 (FN3) domain interacts with DNA and heparin, and plant homeodomain (PHD) mediates binding to histones to regulate transcription . 19,32 The meprin and tumour necrosis factor (TNF) receptor-associated factor homology (MATH) domain interacts with TNF receptors and regulates the function of important transcription factors, such as NF-κB. 34 Less common C-terminal structures include ADPribosylation factor-like (ARF), filamin-type immunoglobulin (FIL), transmembrane (TM), and NHL (named after three proteins, with "H" representing HT2A [TRIM32]) domains. In summary, the high variability of C-terminal domains determines TRIM's specificity and functional diversity by binding to different substrates, thus exerting diverse regulatory effects. 40 2.2 | Functions of TRIM family proteins TRIM proteins share E3 ubiquitin ligase activity; therefore, they participate in the ubiquitin-proteasome pathway, which is an important post-translational modification process for regulating the homeostasis and degradation of proteins. 41 Ubiquitin, a highly conserved protein of 76 residues, is expressed in all eukaryotic cells. 14 Ubiquitination is an ATP-dependent process in which the C-terminal glycine of ubiquitin is covalently bound to a lysine residue of a target protein via the sequential catalysis by an E1 ubiquitinactivating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase. 42 Afterwards, the ubiquitinated targets are subjected to proteasome degradation, wherein the fate of the substrate is determined by the amount of modified lysine and conjugated ubiquitin. 43 Depending on the functions of target proteins, ubiquitination participates in many biological processes, such as autophagy, innate immune signalling pathways (e.g., JAK-STAT3 pathway and TLR-mediated pathway) and carcinogenesis. 14 Most TRIMs function as E3 ubiquitin ligases to regulate these biological processes, which are depended on the specific substrates recognized by them. Moreover, some TRIMs regulate these processes by binding to ubiquitin-like proteins including SUMO 44 and dNEDD8. 45 The nine unclassified TRIM proteins lacking the RING domain may indirectly promote ubiquitination or act in conjunction with other TRIM proteins. 15 As a family characterized by specific structural features rather than functional purposes, TRIM proteins are described as modulators of multiple cellular and physiological activities related to many diseases (e.g., cancer, viral infection and autoimmune disorders) by regulating the activity, stability, degradation, distribution and interaction process of some key proteins. 31,46 T A B L E 1 Classification and structure of TRIM proteins in humans  C-I  TRIM1, TRIM9, TRIM18,  TRIM36, TRIM46, TRIM57,  TRIM67   Interaction with microtubule  cytoskeleton 29   C-II  TRIM54, TRIM55, TRIM63  Muscle protein turnover 30   C-III  TRIM42  Interaction with DNA and  heparin 19   C-IV  TRIM4, TRIM5, TRIM6,  TRIM7, TRIM10, TRIM11,  TRIM15, TRIM17, TRIM21,  TRIM22, TRIM25, TRIM26

| TRIM FAMILY PROTEINS IN IBD
Intestinal barrier dysfunction is an important hallmark of IBD. 47

| Mechanical barrier
The mechanical barrier, also known as the physical barrier, is an essential defensive line of the intestinal barrier. Its purpose is to separate

| Biological barrier
The biological barrier is composed of trillions of microorganisms that colonize the intestine. These microorganisms are referred to as the intestinal microflora and could assist in nutrition absorption, host defence and the function of the immune system. 77 Changes in the impacts on IBD. 78 A recent study reported decreased TRIM31 expression in the intestinal tracts of CD patients. 28 Subsequent experiments revealed that TRIM31 usually accumulates around one type of ubiquitin-coated invasive bacteria, such as Shigella or Salmonella. This is attributed to the specific interaction between TRIM31 and the bacterial receptor NDP52, which induces protective autophagy against invasive bacterial infection or other normal commensal bacteria. 28 Therefore, it was inferred that TRIM31 plays essential roles in restricting invasive bacterial infection in intestinal epithelial cells by promoting autophagy, which can affect gut microbes and lead to IBD in pathological cases. 28,79

| Chemical barrier
The impairment of intestinal chemical barrier, primarily mucus and antimicrobial peptides, can contribute to IBD. 80 The mucus produced by goblet cells, is rich in mucin glycoprotein, and Mucin-2 is one of its most abundant components. 12  whereas the latter involves the processes of humoral and cellular immunity. 85 In the following section, we review the association between the immune barrier and TRIM proteins in the pathogenesis of IBD.

| Innate immunity
Innate immunity serves as the first line of defence against pathogens and acts by mediating intestinal microorganism recognition and quickly inducing inflammatory responses. 85 NOD2, CARD9 and TLR2 are some of the crucial molecules involved in innate microbial sensing.
The NOD2 gene, located in the genomic region imparting IBD susceptibility as determined by genome-wide association studies, encodes NOD2, an intracellular pattern recognition receptor that targets peptidoglycans present in gram-positive or gram-negative bacteria, and subsequently activates the NF-κB signalling pathway. 86 Both TRIM22 and TRIM27 have been identified as regulators of NOD2 and contribute to intestinal inflammation in IBD. 60,61,63 CARD9 is another important component of innate microbial sensing pathways identified by exome sequencing for its involvement in IBD, which also lead to the activation of the NF-κB signalling pathway and cytokine production, especially in fungal infection. 87  In addition to microbial recognition, TRIM proteins also participate in IBD pathogenesis by regulating innate immune cells, such as macrophages and myeloid cells. 85 TRIM33 is essential for monocyte recruitment and differentiation along with macrophage M1/M2 switch and membrane-bound TNF expression, whose impairment may result in an increase in monocyte and decrease in macrophage counts in the colon accompanied by upregulated colonic inflammation. 68 In DSS-induced murine models, TRIM31 was observed to promote the ubiquitin-proteasome pathway of the NLRP3 inflammasome in macrophages, whose defects have been implicated in IBD pathogenesis. 67 3.1.6 | Adaptive immunity TRIM family proteins participate in the cellular immunity process of CD 4+ T lymphocyte differentiation into helper cell types (Th1, Th2 and Th17) and regulatory T (Treg) cells. TRIM21 was observed to have protective roles in IBD by downregulating some pro-inflammatory cytokines (e.g., IL-6 and TNF-α) and inhibiting CD 4+ T cell differentiation into Th1 and Th17 cells. 58,59 The IL-6/JAK/STAT3 signalling pathway has been reported to regulate the differentiation of multiple immune cell types, such as Th2 and Th17 cells. 88 TRIM27 promotes the interaction between STAT3 and JAK1 in this pathway to trigger STAT3 activation in the serum or colonic mucosa, thereby inducing inflammation. 62 TRIM27 knockout mice were observed to gain stronger resistance to DSS-induced colitis, and histopathological examination revealed that their colonic mucosa was more intact, with less disruption. 62 TRIM28 influences the expansion and differentiation of T cells by silencing Tregcharacteristic genes, thus influencing effector, regulatory and helper T cell phenotypes and autoimmunity in the intestinal tract. 64 In addition, a TRIM30α-dependent signalling pathway initiated by the lymphotoxin-β receptor on macrophages was observed to downregulate the inflammatory response during DSS induction. 66 4 | TRIMS AND GENETIC SUSCEPTIBILITY TO IBD TRIM20, encoded by MEFV, is the most-studied member of the TRIM family that contributes to the genetic susceptibility of IBD. [54][55][56][57][89][90][91] Recently, it was reported that TRIM20 not only affects the prevalence of IBD but also influences phenotypic characteristics or complications of IBD. [89][90][91] Several studies have illustrated a correlation between human MEFV mutation and paediatric colitis or IBD, among which IBD complicated with FMF is quite common. [54][55][56][57] Recently, two cases of paediatric IBD were reported in gorillas' model with typical clinical signs, higher levels of C reactive protein and calprotectin, and a clear response to steroids. 92 Sequencing analysis showed that both of them shared a variant of MEFV, which indicates the involvement of TRIM20 in paediatric IBD. 92

| TRIMS AND OPPORTUNISTIC INFECTION IN IBD
A higher risk of severe infections as well as opportunistic infections in IBD patients has been reported, which is generally believed to be a result of the nature of the disease itself or the adverse events associated with the administration of immunosuppressants. 97 Opportunistic infection can aggravate intestinal inflammation and enhance the prevalence of refractory IBD; thus, some opportunistic pathogens can contribute to IBD pathogenesis or progression. 98 Human cytomegalovirus (HCMV), Clostridium difficile (C. diff), Mycobacterium tuberculosis (MTB) and Candida albicans are some of the implicated pathogens, due to their substantial prevalence and incidence. 98 TRIM family proteins have also been shown to be involved in opportunistic infections (Table 3).
HCMV usually erupts during the immunosuppressive period of IBD, which is reportedly induced by the mTOR-mediated TRIM28-related phosphorylation switch. 103  One group reported that TRIM29 mediates the ubiquitination of stimulator of interferon genes (STING) and the STING-TBK1-IRF3 signalling pathway, thereby inhibiting interferon-I and proinflammatory cytokine production, which contributes to the essential roles of TRIM29 in many autoimmune diseases, including IBD. 105 Subsequently, this group tried to develop a novel therapy by targeting the inhibition of TRIM29 via gene silencing for diseases such as IBD. 105 As noted above, TRIM31 downregulates intestinal inflammation in CD by inducing autophagy. 28 Therefore, compounds comprising TRIM31 and its activator were developed by the same team for treating IBD. 106 They also proposed the use of TRIM31 for the diagnosis of IBD or screening of IBD therapeutics. 106 (Table 1), it is difficult to identify specific regularity between TRIM subfamilies and their roles in IBD, despite the structural and functional similarities within each subfamily.
Moreover, most studies evaluating the role of TRIM in IBD have centred on TRIM20-for which retrospective studies usually predominate-while underlying mechanisms remain unclear. Furthermore, the TRIM family is widely accepted as a regulator of tumour oncogenesis or suppression, and its role in colorectal tumours has been reported by several studies. 108 Nevertheless, few studies on TRIM proteins and IBD-related carcinogenesis have been reported, so efforts should be made to explore this critical issue. Further clarification of these questions will enable the development of emerging IBD therapies based on the modulation of TRIM family proteins.

CONFLICT OF INTERESTS
The authors declare that they have no conflicts of interest.

DATA AVAILABILITY STATEMENT
DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article, as no new data were created or analyzed in this study.