TRIM family proteins: emerging class of RING E3 ligases as regulator of NF-κB pathway

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TRIM family proteins; Emerging class of RING E3 ligases as regulator of NF-κB

pathway

Dhanendra Tomar2,3

, Rajesh Singh1, *

Authors Affiliations:

1Department of Biochemistry, Faculty of Science, The M.S. University of Baroda, Vadodara-

390 002, Gujarat, India

Email: [email protected], Tel- +91-9377155303

2Department of Cell Biology, School of Biological Sciences and Biotechnology, Indian

Institute of Advanced Research, Gandhinagar, India

3Present address: EA4576, Maladies Rares: Genetique et Metabolisme (MRGM), University

of Bordeaux, Bordeaux Cedex- 33000, France

Email- [email protected], Tel- +91-9375735047

*Corresponding author

Short Title: TRIM’s mediated regulation of NF-κB

Keywords

NF-κB; Ubiquitin E3 ligases; RING E3 ligase; TRIM proteins; Ubiquitination

mailto:[email protected]

mailto:[email protected]


Abstract

The nuclear factor κB (NF-κB) transcription factor family plays a key role in regulation of

the inflammatory pathway in response to different physiological stimuli starting from

development to ageing. The dysregulation of NF-κB has been associated with many

pathological conditions like inflammatory diseases, neurodegeneration, metabolic diseases

and various kinds of malignancies. The NF-κB pathway is regulated by number of post-

translational modifications, including phosphorylation and ubiquitination. Ubiquitin E3

ligases are key regulators of the process of ubiquitination and provide specificity to the

pathway as they recognize the substrate and determine the topology of ubiquitination.

TRIMs, members of RING family of ubiquitin E3 ligases, are characterized by the presence

of three conserved domains, RING, B-Box, coiled-coil (RBCC). Emerging evidence suggests

that TRIMs regulate innate immune signaling during infection and different pathological

conditions. The studies have demonstrated the role of TRIMs in regulation of inflammatory

pathways including NF-κB. Recent reports suggest that TRIMs play a critical role in

regulation of the NF-κB pathway by ubiquitinating proteins at different steps. In the current

review, we discuss the role of TRIMs as novel NF-κB regulators and their role in different

patho-physiological conditions.


1. Introduction

The patho-physiology associated with numerous chronic inflammatory diseases,

cardiovascular diseases, diabetes and cancers is due to dysregulated immune response. The

altered regulation of the NF-κB pathway has been observed in chronic and acute

inflammatory conditions (DiDonato et al., 2012), hence the mechanism of regulation has

been a major focus of the research in last two decades (Hayden and Ghosh, 2004; Hayden

and Ghosh, 2012; O'Dea and Hoffmann, 2009). This pathway is regulated by several post-

translational modifications, however the ubiquitination plays a key role in regulation of

kinase activity and turnover of the target proteins involved in the pathway (O'Dea and

Hoffmann, 2009). Ubiquitin-mediated post-translational modification is known to regulate

both protein turnover and its function (Haglund and Dikic, 2005). The process of

ubiquitination involves sequential action of three enzymes: E1, E2 and E3. The terminal

enzymes, i.e. ubiquitin E3 ligases, determine the specificity of the pathway by recognizing

the substrate and facilitating the transfer of the Ub (Deshaies and Joazeiro, 2009). E3 ligases

show maximum diversity in higher organisms and more than 1000 have been postulated in

humans. The Ub transferred to the target protein itself can act as substrate for another Ub

moiety, which results in the formation of poly-Ub chains (Deshaies and Joazeiro, 2009). The

poly-Ub chains have a different topology based on the K-residue of substrate Ub involved in

poly-Ub chain formation (Deshaies and Joazeiro, 2009). The E3 ligase and its interaction

with specific E2 enzymes play a critical role in determining the topology of the poly-Ub

chain (Berndsen and Wolberger, 2014; Metzger et al., 2014; van Wijk and Timmers, 2010;

Ye and Rape, 2009). Different types of ubiquitination are involved in regulation of NF-κB

signaling, where a specific ubiquitin E3 ligase is recruited in response to the particular patho-


physiological stimuli. TRIM proteins, members of the RING family of E3 ligases, are well

known for their role in regulation of innate immune response during viral infection (Kawai

and Akira, 2011; Song, 2009). Emerging evidence suggests that TRIMs play a crucial role in

several cellular processes and have been associated with various pathologies like cancer,

inflammatory and autoimmune disorders (Cambiaghi et al., 2012; Micale et al., 2012; Petrera

and Meroni, 2012). Recently, numerous reports have suggested the role of TRIMs in

regulation of the NF-κB pathway. Here, we have reviewed the role of TRIM family proteins

in the regulation of the NF-κB pathway and associated patho-physiological conditions.

2. The NF-κB pathway: Role in different physiological and pathological conditions

Inflammation and its association with different pathological conditions are well established.

NF-κB has been associated with different diseases ranging from infection, neuronal plasticity,

neurodegeneration, cancer, autoimmunity (Wong and Tergaonkar, 2009). NF-κB is activated

by different stimuli such as physical and chemical stresses, microbial products, pro-

inflammatory cytokines, T and B cell mitogens (Hayden and Ghosh, 2004). NF-κB in turn

regulates the expression of many cytokines, chemokines, adhesion molecules, acute phase

proteins and anti-microbial peptides. These molecules regulate immunity, inflammation, cell

proliferation and apoptosis (Hayden and Ghosh, 2004). Hence, it has been one of the most

widely studied pathway in the last decades (Hayden and Ghosh, 2012). The mechanism of

activation of NF-κB in response to different stimuli, yet generating unique outcome, still

needs to be understood.


2.1. NF-κB: Family of transcription factors

The NF-κB family of transcription factors comprises of five members RelA (p65), RelB, c-

Rel, p50, and p52, which form distinct homo- and hetero-dimeric DNA-binding complexes

(O'Dea and Hoffmann, 2009). The well-studied NF-κB dimer is p50/p65, which is in

complex with IB (Inhibitor of kappa B alpha) and is localized in the cytoplasm in resting

stage. The NF-κB-activating signals lead to the phosphorylation, ubiquitination and

subsequent proteasomal degradation of IB in the cytosol. The degradation of IB

exposes the nuclear localization signal (NLS) on p65 (Oeckinghaus and Ghosh, 2009),

induces translocation of p50/p65 to the nucleus where it binds to the κB promoter site of the

target gene (Hayden and Ghosh, 2004).

The NF-κB pathway is activated by ligand-receptor binding at the cell surface or

intracellularly in case of endosomal TLR receptors and RIG-I (Figure 1). The binding of the

ligand to its cognate receptor leads to assembly of the multimeric protein complex on the

cytoplasmic side of the receptor (Figure 1). This complex activates the TAK1/TAB1 kinase

complex after numerous post-translational modifications including phosphorylation and

ubiquitination. Subsequently, the TAK1/TAB1 kinase complex activates the IKK (IκB

kinase) complex, which further phosphorylates IB (NF-kappa-B inhibitor alpha) (Figure

1). The IKK kinase complex is composed of three subunits: IKK, IKK and IKK (NF-κB

essential modulator- NEMO) (Israel, 2010). The various biochemical studies on the purified

IKK complex suggested that the IKK proteins exist in the complex at a 1:1:2 ratio (Hacker

and Karin, 2006; Israel, 2010; Liu et al., 2012). IKK and IKK have kinase activity while

NEMO has a regulatory function in the IKK complex (Hacker and Karin, 2006; Israel, 2010;


Liu et al., 2012). IKK and IKKare homologous and share 51% sequence identity

(Mercurio et al., 1997). Besides the kinase domain, both have a leucine zipper (LZ) domain, a

helix–loop–helix (HLH) domain, and a C-terminus NEMO-binding domain (NBD) (Israel,

2010; Liu et al., 2012).

All the three subunits of IKK complex have distinct functions. IKK knockout mice show

compromised NF-κB activity, while IKK knockout mice show an almost complete lack of

IKK activity and NF-κB activation. Interestingly, IKK has an ubiquitin-like domain (ULD)

just after the kinase domain (May et al., 2004). It may not perform the typical functions of

ubiquitination, however, since the deletion or mutation of the conserved lysine of this domain

decreased kinase activity (Liu et al., 2012). The evidence also suggests the involvement of

the domain in the detachment of IKKβ from p65 following IκBα phosphorylation (Liu et al.,

2012). In contrast to IKK and IKK, NEMO lacks the kinase catalytic domain and is

structurally different from these two proteins. NEMO has two coil-coil domains (CC), a LZ,

and a zinc finger (ZF) domain (Israel, 2010; Liu et al., 2012). The exact mechanism of

NEMO- mediated regulation of the kinase activity of the IKK complex is still not well

understood. NEMO oligomerizes and binds to K63-linked poly-ubiquitin chains via the

ubiquitin binding domain (Uba), which plays a critical role in IKK activation (Chen, 2005;

Chen, 2012; Hayakawa, 2012; Wertz and Dixit, 2010). The studies also suggest that NEMO

interacts with poly-ubiquitinated upstream kinases, such as RIP1 during NF-κB activation

and may recruit other IKKs forming distinct signalosomes leading to the activation of kinase

activity of the IKK complex (Ea et al., 2006; Wu et al., 2006). Phosphorylation of IB by

the IKK complex results in its subsequent ubiquitination and degradation by the proteasome

(Figure 1). This strongly suggests that the ubiquitination has important implications in

regulation at different steps of the NF-κB pathway.


3. The ubiquitination system

Ubiquitination is a most imperative and versatile post-translational modification, playing a

decisive role in the modulation of several signaling cascade events (Haglund and Dikic,

2005). Ubiquitin (Ub) is a highly conserved 76 amino acid globular protein (Figure 2A). The

key features of Ub include its C-terminal di-glycine motif and seven conserved lysine

residues (Figure 2A). The structure of Ub shows a compact globular core and has an extended

C-terminal domain that contains a di-glycine motif. All the lysines of ubiquitin are exposed

on the surface and have the potential to form linkages with other ubiquitin molecules leading

to the diverse topology of poly-Ub chains (Figure 2A). Ub is usually conjugated to the ε-

amino group of lysine residues in substrates. The conjugation of Ub to its target protein is

mediated by the sequential action of three enzymes. The first enzyme in this process is the

Ub-activating enzyme (E1s), which forms a thiol ester bond with the C-terminal carboxyl

group of Ub, thereby activating the C terminus of Ub for nucleophilic attack. The second

enzyme, the Ub-conjugating enzyme (E2s), transiently carries the activated Ub molecule as a

thiol-ester and transfers it to E3 and/or the target protein. The terminal enzyme, Ub-ligases

(E3s), transfers the activated Ub from the E2 to the substrate (or Ub) lysine residue (Figure

2B), resulting in an iso-peptide bond between the substrate lysine and the C-terminus of Ub.

The transfer of Ub to a substrate protein is the key step and thus E3 ligases recognize the

substrate, provide the specificity and regulate the pattern of ubiquitination (Figure 2B).

The diverse types of poly-ubiquitin chain modification of targets can have distinct cellular

functions (Figure 2C). In its simplest form, a single Ub molecule is attached to a single lysine

residue in a substrate, which is defined as mono-ubiquitination (Hicke, 2001). Mono-

ubiquitination is involved in protein trafficking, endocytosis, gene expression, protein-protein

interaction and autophagic degradation of proteins (Chen and Sun, 2009). Alternatively,


several single Ub molecules can be attached to several different lysines, which are referred to

as multiple mono-ubiquitination or multi-ubiquitination (Figure 2C) (Komander and Rape,

2012). Interestingly, all seven lysines (K6, K11, K27, K29, K33, K48 and K63) have the

potential to be used in chain formation, giving rise to chains with different linkages or

branches (Figure 2C) (Komander and Rape, 2012; Pickart and Fushman, 2004). The multi-

ubiquitination of a target protein is generally involved in endocytosis (Chen and Sun, 2009).

K6, K11 and K29 linked poly-ubiquitination of target proteins regulates cell cycle, DNA

repair and proteasomal degradation (Komander and Rape, 2012). K27 linked poly-

ubiquitination is known to be involved in regulation of kinase activity and proteasomal

degradation (Komander and Rape, 2012). The topology of ubiquitin chains formed on the

substrate lysine is decided by E2s and their interaction with E3 enzymes (Berndsen and

Wolberger, 2014; Metzger et al., 2014; van Wijk and Timmers, 2010; Ye and Rape, 2009).

E2 enzymes like UBE2W and UBE2E2 interact with the BRCA1 and BARD1 E3 ligase

complex, respectively and act as ubiquitin chain initiator while UBE2N–UBE2V1 and

UBE2K is involved in chain elongation (Christensen et al., 2007). Few E2 enzymes like yeast

E2 Cdc34 have the capacity to both initiate and elongate Ub chain formation with E3 SCF

(Verma et al., 1997). The various E2 enzymes have the specificity to catalyze the formation

of specific lysine linkages on the substrate. UBE2K, UBE2r1 and UBE2G2 is involved in

formation of K-48 linked Ub chains, the UBE2N–UBE2V1 complex is involved in K-63

linked Ub chain formation whereas UBE2S catalyzes the formation of K-11 linked Ub chains

(Shin et al., 2011; van Wijk and Timmers, 2010).

The ubiquitination of substrate proteins is the key step in the process of protein homeostasis.

K-48 linked ubiquitination of proteins leads to their proteasomal degradation. The

proteasome is a 2.5 MDa sub-cellular system that is required for the degradation of


ubiquitinated proteins and plays a key role in cellular proteastasis. The proteasome contains

two major subunits; the 20S core particle, which is made up of 28 subunits and the 19S

regulatory particle, which is made up of 19 subunits (Finley, 2009; Jung et al., 2009). The

ubiquitinated protein substrate is recognized by the 19S regulatory particle and is translocated

to the 20S core particle where they are degraded. The core particle has a barrel shaped

structure, where a substrate passes through a narrow gated channel and is degraded (Jung et

al., 2009). The proteasome also contains deubiquitinating enzymes on the regulatory subunit,

which is involved in the removal of the ubiquitin tag from the substrate before degradation

(Finley, 2009). The turnover of numerous proteins which play a crucial role in the regulation

of dynamic cellular processes like cell cycle, proliferation, transcription, signaling pathways,

immune response and cell death is high and they are targeted for degradation by proteasomal

system (Konstantinova et al., 2008).

As mentioned above, E3 plays an important role in the terminal step of ubiquitination by

recognizing the substrate and recruiting the charged E2. In the human genome, more than

1000 E3 ligases have been predicted, however, most of them remain uncharacterized. These

Ub E3 ligases are classified into three major families i.e. RING, HECT and U-Box E3

ligases. RING proteins act as scaffold between the Ub-charged E2 and the substrate. This

helps in the transfer of the Ub moiety from E2 to the substrate. Therefore, the RING family

of E3 ligases are not true enzymes, however they help in the transfer of Ub from E2 to the

substrate (Deshaies and Joazeiro, 2009). The HECT proteins form a thioester bond with Ub

before final transfer to the substrate (Deshaies and Joazeiro, 2009). The U-box is similar to

the RING family in its mechanism of Ub transfer, however it differs in active site

conformation. U-box proteins do not have metal-chelating cysteine residues, which is

characteristic of RING proteins (Deshaies and Joazeiro, 2009). The RING family of E3


ligases is the largest family, including more than 600 members, which are further classified

into subfamilies (Deshaies and Joazeiro, 2009). The Ub E3 ligases may attach one or more

Ub polypeptides to lysine residues of the target protein in order to direct degradation,

transport, or function.

4. Hitherto unknown role of ubiquitination in NF-B pathway and possible

consequences

Modification of a target protein by Ub is generally thought a signal for degradation, however,

recent evidence has established that ubiquitination activates several signaling pathways like

internalization of receptors, activation of different kinases and processing of signaling

molecules to active forms (Chen and Sun, 2009). Ubiquitination is a diverse post-translational

modification system, which regulates several steps in the NF-κB pathway starting from the

assembly to the termination of the response (Figure S1). The various types of ubiquitination

mediated through different lysines (K11, K48, K63 and linear/terminal) are involved in NF-

κB regulation (Iwai, 2012). Recent evidence supports an emerging concept that multiple

proteins in the NF-B signaling pathway act as E3 ligases and initiate ubiquitination of

downstream components (Chen and Chen, 2013). The TRAF family members have E3 ligase

activity, and have been shown to be involved in auto-ubiquitination (Habelhah, 2010). Auto-

ubiquitination of TRAF proteins is necessary for activation of the downstream TAK1/TAB1

and IKK kinase complexes (Habelhah, 2010). The IAPs are another class of Ub E3 ligases,

which regulate NF-κB signaling by regulating K-63 linked ubiquitination of RIP1 (Gyrd-

Hansen and Meier, 2010). However, the role of many other RING, HECT and U box domain

Ub ligases as well as putative Ub ligases has not been studied.


4.1. Emerging role of linear poly-ubiquitination in NF-κB activation

Besides the traditional K48 and K63 mediated ubiquitination, recent reports clearly suggest

the crucial role of linear poly-ubiquitination in the regulation of NF-κB pathway (Emmerich

et al., 2011; Iwai and Tokunaga, 2009; Tokunaga, 2013). The traditional poly-ubiquitin chain

is formed through the conjugation of incoming Ub to any of the conserved lysine residues of

the target Ub moiety (Emmerich et al., 2011; Tokunaga and Iwai, 2012b). However, besides

the conserved lysine residues of the Ub, the first methionine (M1) can also acts as acceptor

site for the incoming Ub moiety (Emmerich et al., 2011; Tokunaga and Iwai, 2012a;

Tokunaga and Iwai, 2012b). The linkage between the C-terminal carboxyl group of glycine

and N-terminal -amino group of methionine generates a linear poly-ubiquitin chain. The Ub

E3 ligase complex forming linear poly-ubiquitin chains is known as LUBAC (linear ubiquitin

chain assembly complex) (Iwai and Tokunaga, 2009; Tokunaga, 2013; Tokunaga and Iwai,

2012a; Tokunaga and Iwai, 2012b). The LUBAC is a 600kDa large protein complex and its

three components have been identified todate; HOIL-1L, HOIP and SHARPIN (Tokunaga

and Iwai, 2012b). HOIL-1L (Heme-oxidised iron-responsive element-binding protein 2

ubiquitin ligase-1) is a 58 kDa protein having three conserved domains; UBL (Ubiquitin like

domain), NZF (Npl4- type zinc finger domain) and RBR (RING-In between-RING domain)

(Tokunaga and Iwai, 2012b). HOIP (HOIL-1-interacting protein) is a 123 kDa protein and

contains five distinct domains; two NZF type zinc finger domains, one ZF (zinc finger), one

UBA (ubiquitin associated domain) and one RBR domain (Tokunaga and Iwai, 2012b). The

third component of LUBAC, SHARPIN (SH3 and multiple ankyrin repeat domains protein

associated RBCK1 homology domain-interacting protein) is a 40 kDa protein having UBL

and NZF domains (Tokunaga and Iwai, 2012b). The UBL, UBA and NZF domains of


SHARPIN, HOIL-1L and HOIP are involved in ubiquitin binding and LUBAC formation

(Iwai and Tokunaga, 2009; Tokunaga, 2013; Tokunaga and Iwai, 2012a; Tokunaga and Iwai,

2012b). The ZF domain of HOIP interacts with NEMO and the RBR domain of HOIP is the

active site for the formation of poly-Ub chains (Iwai and Tokunaga, 2009; Tokunaga, 2013;

Tokunaga and Iwai, 2012a; Tokunaga and Iwai, 2012b).

Linear poly-ubiquitination of NEMO is essential for IKK complex assembly as well as its

interaction with other components of the NF-κB signaling cascade (Emmerich et al., 2011;

Iwai and Tokunaga, 2009; Tokunaga, 2013; Tokunaga and Iwai, 2012a; Tokunaga and Iwai,

2012b). The linear poly-Ub chains conjugated to NEMO interact with cIAPs and RIP1 of

TNF receptor complex (Emmerich et al., 2011; Iwai and Tokunaga, 2009; Tokunaga, 2013;

Tokunaga and Iwai, 2012a; Tokunaga and Iwai, 2012b). This interaction of NEMO with the

receptor complex activates the kinase activity of IKK complex, leading to phosphorylation of

IB. The linear poly-Ub chains and LUBAC are now known to regulate varied cellular

events (Emmerich et al., 2011; Iwai and Tokunaga, 2009; Tokunaga, 2013; Tokunaga and

Iwai, 2012a; Tokunaga and Iwai, 2012b). These data suggest that Ub has the potential to form

chains of different types mediated through different types of lysine of the Ub to the substrate,

in turn leading to a discrete outcome in the terms of cellular outcomes. The terminal enzyme

of the ubiquitination, E3 ligase, along with E2 enzymes, plays an essential role in

determining the topology of the ubiquitin chain (Behrends and Harper, 2011). The emerging

classes of different types of E3 ligases perform distinct functions in the different signaling

pathways including NF-κB.


5. TRIM Proteins: Unique Members of RING Ub E3 ligases

The TRIM proteins are unique members of the RING family having a characteristic and

preserved order of the domains. These proteins, also known as RBCC, can be defined as

modular proteins, since they have aRING domain, one or two B-boxes and a Coiled-coil

region (Table 1) (Micale et al., 2012). This RBCC motif is usually followed by either one or

two C-terminal domains, which are specific for each TRIM. The order of the domains is

conserved in TRIMs across various species, which indicates that RBCC motif is the critical

determinant of this superfamily (Ikeda and Inoue, 2012). Some of the TRIMs lack one of the

domains of the RBCC motif, however other domains are still conserved in order as well as

spacing. The first RBCC motif was identified in Xenopus nuclear factor 7 (XNF7) (Reddy et

al., 1991). The other members of TRIM family were identified later, however, the availability

of new genome sequences and functional genomics approaches may lead to identification of

new members (Ota et al., 2004; Reymond et al., 2001; Strausberg et al., 2002).

5.1. RBCC domains: Signature of TRIM proteins

Todate, more than 70 members of TRIM family have been identified in the human genome

and their homologues have also been identified in other eukaryotic species (Consortium.,

1998; Ikeda and Inoue, 2012; Marin, 2012; Meroni, 2012; Micale et al., 2012; Miyamoto et

al., 2002). However, among the large number of identified TRIM proteins, only a few have

been well characterized so far. TRIM family proteins are distinct from each other in the

number of B-boxes and the nature of the C-terminal domain. The RING domain of TRIM

proteins is a specialized zinc finger of 40–60 residues, located within 10–20 amino acids

from the N-terminus of nearly all TRIMs, which binds to two zinc atoms (Micale et al.,


2012). The RING domain was initially predicted to be involved in protein-protein

interactions (Borden, 2000; Lovering et al., 1993), however, now most of them are shown to

possess Ub E3 ligase activity (Freemont, 2000; Joazeiro and Weissman, 2000). E3 ligase

activity may be a common feature of all TRIM proteins, although this has yet to be

investigated fully. Some of the TRIM family proteins are also known to regulate ubiquitin

like protein modifications like SUMOylation and ISGylation (Chu and Yang, 2011;

Napolitano and Meroni, 2012).

The RING domain in the TRIM protein is generally followed by B-box domains; B1 and B2.

The B-boxes have two 40 residueslarge CHC3H2 zinc-finger motifs (Reddy et al., 1992). The

B1 and B2 B-boxes differ from each other in number and spacing of the conserved cysteine

and histidine residues (Borden et al., 1996). The B1 box always precedes B2, whenever both

B1 and B2 domains are present whereas only B2 may be found alone (Borden et al., 1996).

Interestingly, B-boxes are found exclusively in TRIM proteins and are probably an important

determinant of the family, but their functions have not been studied in detail.

The B-box domains are usually followed by a coiled-coil (CC) domain, which is the third

signature sequence, a typical hyper-secondary structure formed by the intertwining of

multiple α-helices. The CC domain can interact with a CC domain of the same or of other

TRIM proteins and hence can mediate both homo- and hetero-oligomeric interactions (Li et

al., 2011b). It is also becoming evident that other domains of TRIM proteins can also

influence these interactions. The B-box domain of TRIM27 and the whole RBCC region of

TRIM28 are reported to be essential for the formation of CC-domain mediated multimeric

protein complexes (Cao et al., 1997; Peng et al., 2000). The CC-domains mediated protein–

protein interactions forms higher order structures (Peng et al., 2000; Reymond et al., 2001).

The established example of CC domains mediated high-molecular-mass protein complexes


formation is assembly of PML nuclear bodies (Bernardi and Pandolfi, 2007). These nuclear

bodies are composed of the SUMOylated scaffolding protein PML with numerous other

proteins, which have a critical role in various cellular processes (Bernardi and Pandolfi,

2007). The CC-domain, among other functions, also defines specific sub-cellular localization

(Peng et al., 2000; Reymond et al., 2001).

5.2. C-terminal domains: Sub-families of TRIM proteins

The RBCC motif of TRIM family proteins is usually followed by one or two C-terminal

domains of various length and composition. The TRIM family can be classified into eleven

sub-classes based on domains present in the C-terminal region (Kawai and Akira, 2011;

Ozato et al., 2008). The C-terminal region of TRIM proteins is reported to have any of 10

distinct motifs (Table 1) (Kawai and Akira, 2011; Ozato et al., 2008). PRY and SPRY

domains are most commonly found in TRIM proteins (Grutter et al., 2006; Woo et al., 2006).

This constitutes the biggest family of the TRIM proteins. There are approximately 33 TRIMs

out of a total 100 proteins containing PRY-SPRY domain in the human genome (Table 1).

TRIM proteins having a PRY-SPRY domain are important in the innate immune response

(D'Cruz et al., 2013; Perfetto et al., 2013). TRIM5 and TRIM21 are well known PRY-SPRY

domain-containing proteins and are involved in the antiviral response which will be discussed

further.

The other group of TRIMs have a COS domain (C-terminal subgroup one signature) along

with the PRY-SPRY domain. Generally, the COS domain is found downstream of the CC

domain in a few of TRIM family members (Table 1). The COS domain is also present in

several non-TRIM proteins (Short and Cox, 2006). The group of proteins having the

signature motif of COS- PRY-SPRY and FN3 constitute the second largest group (Table 1).


The proteins of this family regulate cell cycle (TRIM36) and neuronal functions (TRIM9)

(Miyajima et al., 2009; Tanji et al., 2010). TRIM42 is the only protein which has the typical

C-terminal motif of COS-FN3, however its cellular functions remain uncharacterized (Table

1). The other sub-family of TRIM protein has the terminal signature of a COS-ACID domain

(Table 1). These proteins have a glutamic acid rich region and are specifically expressed in

muscles and are developmentally regulated (Perera et al., 2012). They play an important role

in regulation of autophagy in muscle cells (Perera et al., 2012). PHD (plant homeodomain)

domain-containing proteins have been classified as epigenome reader (Sanchez and Zhou,

2011). There are three members having the PHD signature: TRIM24, TRIM28 and TRIM33

(Table 1) (Herquel et al., 2011). These proteins are nuclear proteins and are known to form

multimeric complexes for chromatin remodeling and initiation of transcription.

There are two subgroups of proteins which contain a FIL (filamin-type immunoglobulin)

domain (Table 1). TRIM45 only has a FIL domain at the C terminus. and can regulate NF-κB

and the PKC mediated signaling pathway (Sato et al., 2014; Shibata et al., 2012). The other

sub-group of proteins contains an NHL domain (NCL1, HT2A and LIN41 domain) along

with the FIL domain (Table 1). All other subfamilies generally have one or two

representative TRIM proteins with characteristic domains. TRIM37 contains a characteristic

MATH (meprin and TRAF-homology) domain (Table 1). This suggests that the MATH

domain containing protein has a characteristic property of self olgomerization. The cellular

function of TRIM37 is not well understood, however mutation leads to Mulibrey Nanism as a

new peroxisomal disorder (Hamalainen et al., 2004).

TRIM23 has an ARF (ADP-ribosylation factor) domain at the C terminus (Table 1) and its

role is emerging in innate immune response and will be discussed later in the review (Kawai

and Akira, 2011; Ozato et al., 2008). Interestingly, two TRIM proteins have a transmembrane


domain at the C-terminus (TRIM13 and TRIM59) (Table 1). TRIM13 is localized to ER and

plays an important role in autophagy induction and caspase activation during ER stress

(Tomar et al., 2013; Tomar et al., 2012a). TRIM59 localization and function still remains

uncharacterized. There is another sub-group of TRIM proteins which does not have any

characteristic C-terminal domain. There are more than 8 proteins in this group and the

characteristic domain for this group of proteins needs to be defined. TRIM19 is well

characterized and plays important role in PML bodies and in regulation of NF-κB. The other

members also show an important role in regulation of inflammatory pathways (Kawai and

Akira, 2011; Ozato et al., 2008). There are several uncategorized TRIMs, which do not have

a characteristic RING domain but have characteristic domains of the RBCC family (Table 1).

The presence of diverse domains in TRIM proteins clearly suggest their role in diverse

cellular processes but until now only few of them have been well studied.

5.3. Relationship between TRIM proteins’ domain organization and functions

As described in the previous section, TRIM proteins have a conserved set of an N-terminal

RBCC domain and diverse types of C-terminal domains. Each domain has a characteristic

function which is also regulated by the presence of the other domains. The RING domain was

initially predicted to be involved in protein-protein interactions (Borden, 2000; Lovering et

al., 1993), however, now most of proteins having RING domain have shown to exhibit E3

ligase activity (Freemont, 2000; Joazeiro and Weissman, 2000). E3 ligase activity of different

TRIMs needs to be systematically investigated. Some of the TRIMs regulate protein

modifications other than ubiquitin like SUMOylation and ISGylation (Chu and Yang, 2011;

Napolitano and Meroni, 2012). Recent reports suggest that the B-box domain of certain

TRIMs also have ubiquitin E3 ligase activity (Bell et al., 2012). However, it is generally


involved in protein-protein interactions and due to its location near to the RING domain, it

may act as substrate recognizing domain for the E3 ligase activity. This evidence suggests

that the interaction of different domains and outcome of its activity needs further study.

The C-terminal domains of TRIMs also have diverse signaling functions. PRY-SPRY

domains are involved in formation of a multimeric signaling complex by interacting with

PRY-SPRY domain of other proteins (Grutter et al., 2006; Woo et al., 2006). Generally,

PRY-SPRY domain containing proteins play a crucial role in immune regulatory pathways.

The COS domain binds to microtubules (Bernardi and Pandolfi, 2007), which may be

involved in cytoplasmic translocation of multimeric signaling complexes formed by TRIMs.

FN3 (fibronectin type 3), a 100 amino acid domain, binds to DNA and heparin binding sites

as well as to the cytoplasmic side of the receptors. PHD domain containing proteins

recognize the epigenetic modification of histone proteins, specifically acetylation and

methylation of arginine and lysine respectively (Herquel et al., 2011). These proteins interact

with other nuclear receptors and modulate their stability through ubiquitination (Herquel et

al., 2011). FIL domain containing proteins have a characteristic actin binding motif and are

involved in many processes like cell morphology, migration and cell signaling (Kawai and

Akira, 2011; Ozato et al., 2008). The NHL domain has recently been shown to bind RNA.

Emerging evidence suggests their role in miRNA biogenesis and translation (Schwamborn et

al., 2009). These data suggest that TRIM proteins can be recruited during different cellular

processes however only few of them have been studied in detail.


5.4. TRIM Proteins: Role in immunity and inflammation

TRIM family proteins are critical regulators of innate immune response specifically in

antiviral immunity (Fletcher and Towers, 2013; Kawai and Akira, 2011). Various studies

suggest that TRIMs regulate/restrict viral infections such as human immunodeficiency virus

type 1 (HIV-1) and N-tropic murine leukemia virus (MLV) influenza virus, vesicular

stomatitis virus (VSV), herpes simplex virus, cytomegalovirus, and HIV-1 (Nakayama and

Shioda, 2010; Ozato et al., 2008; Rahm and Telenti, 2012; Uchil et al., 2008; Versteeg et al.,

2013). TRIM5 is a well characterized member due to its role in anti-HIV response during

its infection (Nakayama and Shioda, 2010; Song, 2009). TRIM5 suppresses viral uncoating

in the cytoplasm of infected cells (Nakayama and Shioda, 2010; Song, 2009). The entry of a

virus into the host cell is promoted by TRIM15 and TRIM38 (Brass et al., 2008; Ozato et al.,

2008; Rahm and Telenti, 2012). On the other hand, TRIM8, TRIM11, TRIM31 and TRIM 21

inhibit viral entry (Brass et al., 2008; Ozato et al., 2008; Rahm and Telenti, 2012). TRIM11

and TRIM32 are known to suppress viral gene expression in host system (Brass et al., 2008;

Ozato et al., 2008; Rahm and Telenti, 2012). TRIM22 and TRIM15 suppress viral assembly

in the host system (Barr et al., 2008; Brass et al., 2008; Ozato et al., 2008). TRIM25,

TRIM31, TRIM62 promote the viral release from the host cell, whereas TRIM11, TRIM26,

TRIM32, TRIM14, TRIM21 and TRIM19 suppress viral particle release from the host cell

(Brass et al., 2008; Ozato et al., 2008; Rahm and Telenti, 2012; Uchil et al., 2013; Uchil et

al., 2008). These reports clearly indicate that TRIM proteins act both suppressor and

promoter of viral infection.


The interferon- and NF-κB- are the critical regulatory pathways in the regulation of innate

immune response during viral infection (Hanada and Yoshimura, 2002). TRIM family

proteins are differentially regulated by interferon (Rajsbaum et al., 2008) and in turn regulate

interferon mediated downstream pathways (Kawai and Akira, 2011). TRIM8 interacts with

SOCS1 which ubiquitinates and promotes its proteasomal degradation and positively

regulates IFNγ mediated signaling (Toniato et al., 2002). TRIM25 positively regulates RIG-1

mediated type-I IFN production by regulating ubiquitination of the same (Gack et al., 2007).

TRIM25 also regulates ISGylation of the RIG-1 complex, which may negatively regulate

interferon signaling, suggesting that TRIM25 may be a critical modulator of IFN signaling

(Kim et al., 2008). The role of other TRIMs in regulation of IFN signaling is also emerging.

Recently, another member of the TRIM family, TRIM4, regulates K-63 linked ubiquitination

of RIG-1 (Yan et al., 2014) and positively regulates type-1 interferon signaling during viral

infection (Yan et al., 2014). TRIM21 (Ro52, SS-A) is a well-known auto-antigen associated

with Sjögren’s syndrome and systemic lupus erythematosus (SLE) (Ben-Chetrit et al., 1988).

TRIM21 interacts with IRF8 (interferon associated transcription factor), in response to IFN

and TLR receptor stimulation (Kong et al., 2007). The ubiquitination of IRF8 in the nucleus

positively regulates the expression of targeted cytokines (Ben-Neriah, 2002). The stimulation

of cells with interferon leads to the accumulation of TRIM21 in the nucleus (Strandberg et

al., 2008) which ubiquitinates IRF8 and enhances cytokine expression, specifically type I

interferon (Oke and Wahren-Herlenius, 2012). These reports clearly indicate that TRIM

family proteins play a critical role in the antiviral signaling and interferon production. The

activation of antiviral signaling is regulated by the IRF family of proteins and they co-operate

with the NF-κB family of the transcription factors (Pfeffer, 2011; Yarilina and Ivashkiv,

2010). It has been observed that along with IFN activation, genes regulated by NF-κB are

also up regulated (Hiscott et al., 2003; Pfeffer, 2011). These genes play an important role in


the innate immune response as well as in other cellular responses like cell survival and death

(Baker et al., 2011; Caamano and Hunter, 2002; Wong and Tergaonkar, 2009). This evidence

strongly suggests that TRIMs regulating innate immune response during viral infection may

play an important role in regulation of NF-κB as well as its cross talk with other

inflammatory pathway.

5.5. TRIMs: Emerging regulator of NF-κB pathway

One of the major arms of innate immunity and inflammation is activation of NF-κB

signaling. NF-κB regulates the expression of various cytokines and other genes regulating

immune functions. NF-κB signaling is activated by various cell surface or intracellular

receptors. The expression of members of the TRIM family proteins is also regulated by NF-

κB regulatory cytokines. The pro-inflammatory cytokine, TNF, regulates NF-κB signaling

under many physiological and pathological conditions. The expression of TRIM9, TRIM21

and TRIM62 is up-regulated in the presence of TNF and may act as positive or negative

regulator of the NF-κB pathway (dos Santos et al., 2004; Schwamborn et al., 2003). The

following sections describe further details about their role in NF-κB pathway.

5.5.1. TRIMs: Positive regulator of NF-κB signaling

The NF-κB pathway is activated in response to numerous stimuli such as cytokines (TNF and

IL1), infection (LPS, diacyl and triacyl lipoproteins, flagellin, ssRNA, dsRNA), and growth

factors. Existing reports suggest that four members of TRIM family proteins (TRIM8,

TRIM20, TRIM23 and TRIM25) positively regulate NF-κB signaling in response to different

cytokines and viral infection. The expression of TRIM22 activates NF-κB in a human

macrophage cell line (Yu et al., 2011) however, another report suggest it suppresses TRAF6

mediated NF-κB activation (Figure 3, table 2) (Qiu et al., 2013). TRIM38 acts as both


positive and negative regulator of NF-κB pathway (Figure 3, table 2). The overexpression of

TRIM38 in HEK293 cells suppresses NF-κB activity (Liu et al., 2011). TRIM8 positively

regulates TNF induced NF-κB activation and acts at two different steps in the NF-κB

pathway (Tomar et al., 2012b). TRIM8 mediated positive regulation of NF-κB is mediated

through the K-63 linked poly-ubiquitination of TAK1 in response to TNF and IL1 (Li et

al., 2011a). The K-63 linked poly-ubiquitination of TAK1 activates the TAK1/TAB1 kinase

complex, which subsequently phosphorylates the IKK leading to the activation of the IKK

kinase complex (Israel, 2010; Liu et al., 2012). The other study reported that TRIM8 is a

nuclear protein and regulates PIAS3 K-48 linked ubiquitination in the nucleus and its

translocation and proteasomal degradation to regulate NF-κB (Tomar et al., 2012b).

Interestingly, TRIM8 translocates to the cytoplasm in response to TNF where it induces K-63

linked ploy-ubiquitination of TAK1 (Tomar et al., 2012b). TRIM20 also known as MEFV

(Mediterranean Fever) protein, regulates the turnover of IB (Inhibitor of kappa B alpha)

and interaction with the p65 subunit of NF-κB, hence positively regulates the NF-κB pathway

(Chae et al., 2008). TRIM20 is a target of caspase-1 and its cleaved N-terminal fragment

binds to the p65 subunit of NF-κB and facilitates its nuclear translocation (Chae et al., 2008).

Interestingly, the N-terminal fragment of TRIM20 also binds to IB and regulates its

calpain mediated degradation (Chae et al., 2008). TRIM20 is known to be mutated in familial

Mediterranean fever. The mutated form of TRIM20 activates caspase-1, therefore its

cleavage and subsequent NF-κB activation (Chae et al., 2008). TRIM23 interacts with human

cytomegalovirus (HCMV) genome encoded UL144 and regulates its interaction with TRAF6

to positively regulate NF-κB signaling (Poole et al., 2009). Another report suggests that

TRIM23 induces K-27 linked poly-ubiquitination of NEMO and positively regulates NF-κB

(Arimoto et al., 2010).


TRIM25 regulates K-63 linked poly-ubiquitination of RIG-I (receptor of anti-viral signaling

cascade) which subsequently positively regulates NF-κB and interferon signaling (Gack et

al., 2007). TRIM4 mediated K-63 linked poly-ubiquitination of RIG-1 positively regulates

both NF-κB and interferon signaling during viral infection (Yan et al., 2014). The evidence

suggests that TRIMs can activate NF-κB in response to different stimuli. However, the

systematic regulation of TRIMs in response to each stimulus is not well understood.

5.5.2. TRIMs: Potential suppressor of NF-κB signaling

There are many reports which suggest that TRIMs fine tunes the response of the NF-κB

pathway; they are summarized in Figure 3 and Table 2. TRIM9 (expressed in brain) interacts

with β-TrCP (a component of Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase

complex), prevents its binding with IκBα and p100 (Shi et al., 2014) and its degradation and

subsequent NF-κB activation (Shi et al., 2014). TRIM19 (promyelocytic leukemia protein-

PML) sequesters NF-κB in nucleus to the PML-nuclear bodies in the presence of TNF and

acts as transcriptional repressor of NF-κB (Wu et al., 2003). TRIM21 is a known auto-antigen

and is involved in regulation of interferon signaling. The auto-antibodies are generated

against TRIM21/Ro52 in patients with lupus and Sjogrens syndrome. Auto-antibodies

generated against TRIM21 prevent E3 ligase activity by inhibiting its interaction with the

cognate E2 (UBE2E1) (Espinosa et al., 2011). In HTLV1 infected cells, Tax oncoprotein

induces persistent phosphorylation and activation of IKK, leading to NF-B activation,

which promotes cell cycle entry and cell survival. Interestingly, TRIM21 inhibits Tax

oncoprotein overexpression induced NF-B activation. TRIM21 induces mono-ubiquitination

and subsequent autophagic degradation of IKK(Niida et al., 2010). This autophagic

degradation of IKK suppresses NF-κB activity (Niida et al., 2010). The other member of

TRIM family, TRIM27, interacts with and suppresses IKK complex activity leading to


suppression of NF-κB in response to TNF, IL-1, TLR3 ligand and viral infections (Zha et al.,

2006). TRIM30a, interacts with the TAB2-TAB3-TAK1 adaptor-kinase complex, which

mediates its endocytic-lysosomal degradation, leading to the suppression of TLR induced

NF-κB activation (Shi et al., 2008). TRIM40 suppresses the NF-κB pathway by regulating

NEMO neddylation (Noguchi et al., 2011). TRIM40 is highly expressed in the

gastrointestinal tract and tumor associated inflammation in the gastrointestinal tract (Noguchi

et al., 2011), and thus may be a potential oncogene linking inflammation and cancer. TRIM45

inhibits TNF induced NF-κB signaling but its molecular mechanism is still not understood

(Shibata et al., 2012). TRIM59 interacts with evolutionarily conserved signaling intermediate

in Toll pathways (ECSIT) and negatively regulates NF-κB signaling in response to RIG-I

pathway activation (Kondo et al., 2012). Our ongoing work on the role of TRIM’s in

regulation of inflammatory pathway shows that TRIM13 interacts and ubiquitinates NEMO.

TRIM13 mediated NEMO ubiquitination leads to suppression of NF-κB signaling (Tomar

and Singh, 2014).

The two members of TRIM family protein (TRIM22 and TRIM38) have a dual role in NF-κB

signaling. TRIM22 suppresses TRAF6 auto-ubiquitination and induces TAB2 degradation

leading to suppression of NF-κB activation (Qiu et al., 2013). The overexpression of TRIM38

positively regulates NF-κB, however the regulatory mechanisms still remains to be

understood (Liu et al., 2011). The expression of TRIM38 is regulated by TLR induced NF-κB

and it induces K-48 linked poly-ubiquitination and proteasomal degradation of TRAF6 in

macrophages in the presence of TLR ligand (Zhao et al., 2012). TRIM38 acts as a feedback

regulatory mechanism to prevent the increased activation of TLR induced NF-κB and

inflammatory response. A recent report suggests that TRIM38 regulates lysosomal

degradation of TAB2/3 to suppress the TNF- and IL-1 induced NF-κB pathway (Hu et al.,


2014). These data suggest that positive/negative regulation of NF-κB by TRIMs may have

important implications in many physiological and pathological conditions.

6. Potential applications of TRIM mediated regulation of NF-κB in disease

management

TRIM mediated regulation of the NF-κB pathway plays a crucial role in the process of

inflammation, immunity and cell signaling. The NF-κB pathway is a potential drug target for

the management of various diseases like cancer, inflammation associated pathologies and

autoimmune disorders (Niederberger and Geisslinger, 2008; Perkins, 2012). The main

objective to target NF-κB in cancer therapy is to inhibit its activity, in turn inhibiting the anti-

apoptotic and other tumor promoting functions. The drug ‘Bortezomib’ a proteasomal

inhibitor, blocks IB degradation and suppresses NF-κB. It is successfully used for the

treatment of haematological cancers (Baud and Karin, 2009; Gilmore and Herscovitch, 2006)

however have met with limited success in complex solid tumors. Many natural product-

based drugs, such as sesquiterpene lactones, are also used for the management of different

cancers (Gilmore and Herscovitch, 2006). Sesquiterpene lactones directly inhibit the binding

of NF-κB to the κB site of the DNA (Gilmore and Herscovitch, 2006). Nonsteroidal anti-

inflammatory drugs (NSAIDs) are the prime drugs used for the treatment of inflammation

associated pathologies. Some of the NSAIDs like ‘aspirin’ and ‘sodium salicylate’ are known

to inhibit the NF-κB pathway by targeting the ATP binding site on IKK (Yin et al., 1998).

The suppression of NF-κB activity of these NSAIDs was also hypothesized to contribute in

their analgesic and anti-inflammatory properties. Glucocorticoids, used for the treatment of

allergies and autoimmune diseases, were also shown to inhibit NF-κB activity (Auphan et al.,

1995).


There are numerous NF-κB regulators which target various steps of the NF-κB pathway

(Gilmore and Herscovitch, 2006), however unselective and complete inhibition of NF-κB

might lead to several serious side effects. Most of the current drugs used in disease

management, which target NF-κB, have adverse side effects. The NF-κB pathway regulates

numerous physiological processes and its complete inhibition may lead to profound

consequences. Therefore, there is a need to understand the modulators of NF-κB in specific

patho-physiological conditions for selective modulation. TRIMs seem to fine tune the NF-κB

signaling. These proteins can form higher-order structures as well as signalosomes in specific

patho-physiological conditions, and hence can be potent targets.

7. Conclusions

The discussed reports clearly indicate that TRIM family proteins play a crucial role in

regulation of NF-κB signaling. The emerging evidence suggests that these proteins can be

classified as modulators of signaling pathways in different conditions (Ozato et al., 2008).

There are more than 70 members in this family while only few members of TRIM family

proteins have been shown to regulate NF-κB signaling (Table 2). TRIMs have multiple

domains and each domain has unique functions in the assembly of signalosomes in response

to a distinct stimulus. The presence of a RING domain suggests that the E3 ligase property is

inherent and plays an important role in modulation of different cellular processes (Ikeda and

Inoue, 2012). It has been observed that these proteins can auto-ubiquitinate under normal

physiological conditions and regulate their own turnover and may be stabilized in the given

stimuli (Ikeda and Inoue, 2012; Ozato et al., 2008; Streich et al., 2013; Tomar et al., 2012a).

It is important to initiate the systematic study of turnover of these proteins in given patho-

physiological conditions to understand its implication in different cellular processes including

NF-κB. B-Box domains have unique characteristics, can bind to Zinc and can act as E3 ligase


domain. TRIMs can homo/heterodimerize with other proteins via the CC domain, hence their

interaction within the other proteins of this family may be important in a given physiological

condition. Similarly, the domain at the C terminus also can bind to a ligand hence they can

respond to a wide variety of ligands and stimuli, which may regulate NF-κB and other

inflammatory pathways. The role of TRIM’s mediated NF-κB regulation in associated

pathological conditions also needs to be understood.

The emerging interest in this group of proteins clearly reflects its importance in the innate

immune response, however their properties should be studied beyond their role in innate

immunity. TRIM32 having an NHL seems to play an important role in regulation of miRNA

biogenesis through turnover of Ago proteins (Schwamborn et al., 2009). miRNAs are a novel

class of regulators of NF-κB and other cellular pathways. The role of TRIM32 is important

for the asymmetric division of the neuronal stem cell suggesting its important role in other

cellular processes (Hillje et al., 2013). Studies on TRIM family proteins have shown that

some members of this family are also involved in vesicular trafficking. TRIM72 is involved

in vesicular trafficking during membrane repair (Cai et al., 2009). Similarly TRIM3 is

involved in ubiquitin mediated endosomal switch of EGFR receptor sorting (Mosesson et al.,

2009). As TRIMs play important roles in various physiological conditions, mutations in these

proteins lead to the onset of various pathological conditions. Mutations in TRIM20, TRIM18,

TRIM32, TRIM37 are associated with familial Mediterranean fever, X-linked Opitz G/BBB

syndrome, Limb-girdle muscular dystrophy type 2H, Mulibrey nanism respectively (Avela et

al., 2000; French, 1997; Frosk et al., 2002; Quaderi et al., 1997). A recent report and study

from our lab strongly suggesst that these proteins can play crucial role in the regulation of

selective autophagy (Mandell et al., 2014; Tomar et al., 2013; Tomar et al., 2012a). Given the

role of autophagy in tumor metabolism, TRIMs can further act as potential oncogene/tumor


suppressors. This hypothesis is further supported as some of the TRIM genes are

homozygously deleted in some cancer conditions, e.g. TRIM13 is deleted B-cell chronic

lymphocytic leukemia (CLL) and multiple myeloma (MM) (Kapanadze et al., 1998).

Further studies will help us to understand the role of TRIMs mediated regulation of NF-κB

and other inflammatory pathways and their implications in different physio/pathological

conditions.

Acknowledgement

The current research work on TRIM proteins is financially supported by the Department of

Biotechnology, Government of India (grant number BT/PR13924/BRB/10/794/2010 to

Rajesh Singh). Authors acknowledge the research fellowship from Council of Scientific and

Industrial Research (CSIR), Government of India and the Indo-French Centre for the

Promotion of Advanced Research (IFCPAR), New Delhi to Dhanendra Tomar.

Conflict of interest

Authors declare there is no conflict of interest.


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Figure 1. The mechanism of NF-κB signaling

The ligands bind to their corresponding receptors on the cell surface or intracellularly and

induces their oligomerization. This receptor-ligand interaction forms intracellular multi-meric

protein complexes at the membrane. These protein complexes subsequently induce

translocation of NF-κB to the nucleus for transcription regulation in the presence of given

stimuli.


Figure 2. The process and diverse outcomes of ubiquitination

(A) Crystal structure of Ub showing seven conserved lysine and C-terminal di-glycine

motif. (B) Ubiquitination of target protein involves the sequential action of three

enzymes: E1, E2 and E3. (C) Differences between mechanism of Ub transfer from E2

to substrate by RING and HECT Ub E3 ligases. (D) Different forms of protein

ubiquitination and their fate. Proteins can be mono-ubiquitinated with a single Ub,

multi-ubiquitinated or polyubiquitinated. Poly-ubiquitination can be of different types

depending on the Ub lysine residue which is targeted for Ub chain elongation. The

different fates of the modified proteins have been shown in the figure.



Figure 3. Role of TRIMs in NF-κB signaling

The numerous TRIM proteins are reported to regulate NF-κB signaling. The reports showed

that TRIM4, 8, 20, 22, 23, 25 and 38 positively regulate NF-κB while the TRIM9, 13, 19, 21,

22, 30a, 38, 40, 45 and 59 negatively regulate NF-κB.


Table 1: Classification of TRIM family proteins based on C-terminal region

TRIM family proteins are categorized into eleven sub-families (C-I to C-XI) based on the

presence of C-terminal domains and one uncategorized (UC) group.


Table 2: TRIM proteins: Regulator of NF-κB signaling: The list and the role of TRIMs,

which have been experimentally confirmed to regulate the NF-κB pathway.

S.No. TRIM Reference

Effect on

NF-κB

Mechanism

1. TRIM4 (Yan et. al. 2014) Positive K63 linked RIG-I poly-ubiquitination

2 TRIM8

(Li et.al.2011) Positive K63 linked TAK1 poly-ubiquitination

(Tomar et.al.2012) Positive

Degrades PIAS3 and increase p65 nuclear

localization, Translocates to cytoplasm in

response to TNF

3 TRIM9 (Shi et al., 2014) Negative

Block IB degradation by interacting with

β-TrCP SCF complex

4 TRIM13

(Tomar and Singh,

2014)

Negative NEMO poly-ubiquitination

5 TRIM19 (Wu et.al. 2003) Negative

Recruitment of NF-κB to promyelocytic

leukemia protein-nuclear bodies (PML-NBs)

6 TRIM20 (Chae et.al. 2008) Positive

Interacts with p65 subunit of NF-κB and

induces calpain mediated degradation of

IB

7 TRIM21 (Niida et.al. 2010) Negative IKK autophagic degradation

8 TRIM22

(Yu et.al. 2011) Positive

Overexpression activates NF-κB, mechanism

unknown

(Qiu et.al. 2013) Negative

Suppress TRAF6 auto-ubiquitination, TAB2

degradation

9 TRIM23 (Poole et.al. 2009) Positive Interacts with Human cytomegalovirus


(HCMV) product UL144 and regulates its

interaction with TRAF6

(Arimoto et.al. 2010) Positive K27 linked NEMO poly-ubiquitination

10 TRIM25 (Gack et.al. 2007) Positive K63 linked RIG-I poly-ubiquitination

11 TRIM27 (Zha et.al. 2006) Negative Interacts and suppress IKKs

12 TRIM30a (Shi et.al. 2008) Negative

Interacts with TAB2-TAB3-TAK1,

Endocytic-Lysosomal degradation of

TAB2/3

13 TRIM38

(Liu et.al. 2011) Positive

Overexpression activates NF-κB, mechanism

unknown

(Zhao et.al. 2012) Negative

K48 linked poly-ubiquitination and

degradation of TRAF6

14 TRIM40

(Noguchi et.al.

2011)

Negative Neddylation of NEMO

15 TRIM45 (Shibata et.al. 2012) Negative

Overexpression suppress NF-κB, mechanism

unknown

16 TRIM59 (Kondo et.al. 2012) Negative

Overexpression suppress NF-κB, interacts

with ECSIT

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TRIM family proteins: emerging class of RING E3 ligases as regulator of NF-κB pathway