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    Topic review

    RhoH in TCR Signalling

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    Definition

    As an atypical member of the Rho family small GTPases, RhoH shares less than 50% sequence similarity with other members, and its expression is commonly observed in the haematopoietic lineage. To date, RhoH function was observed in regulating T cell receptor signalling, and less is known in other haematopoietic cells. Its activation may not rely on the standard GDP/GTP cycling of small G proteins and is thought to be constitutively active because critical amino acids involved in GTP hydrolysis are absent. Alternatively, its activation can be regulated by other types of regulation, including lysosomal degradation, somatic mutation and transcriptional repressor, which also results in an altered protein expression. Aberrant protein expression of RhoH has been implicated not only in B cell malignancies but also in immune-related diseases, such as primary immunodeficiencies, systemic lupus erythematosus and psoriasis, wherein its involvement may provide the link between immune-related diseases and cancer.

    1. Introduction

    The Rho (Ras homologous) family of small GTPases is one of the members of the Ras superfamily. In humans, 20 Rho-family GTPase members have been identified and categorised into eight major subfamilies Rho-, Rac-, Cdc42-, RhoU/RhoV-(also known as Wrch/Wrch2), Rnd-, RhoD/RhoF-, RhoBTB- and RhoH subfamilies. The subfamilies are categorised based on the similarities in their primary amino acid sequence (Table 1), structural motifs and biological functions [1]. Initially, Miro and RhoBTB3 were included as members of the Rho family small GTPases, but they have been excluded due to the lack of similarity between these proteins and other members of the Rho-family. For instance, Miro subfamily proteins lack the Rho insert region and are assumed to be non-catalytic as the sequence in the highly conserved G2–G5 loops responsible for nucleotide binding and hydrolysis is different compared with that in canonical GTPase domains [2][3]. RhoBTB3 shares 25% sequence similarity with and contains the same BTB domains as RhoBTB1 and RhoBTB2. However, this protein is not considered as a Rho GTPase by some studies due to its lack of similarity with Rho and Ras [3][4].
    Table 1. The percentage sequence similarity shared between Rho-family GTPases.
     

    Rho

    BTB1

    Rho

    BTB2

    RhoH

    Rnd1

    Rnd2

    Rnd3

    RhoD

    RhoF

    RhoA

    RhoC

    RhoB

    Wrch2

    Wrch1

    TC10

    TCL

    Cdc42

    RhoG

    Rac2

    Rac1

    Rac3

    RhoBTB1

     

    70

    34

    33

    31

    30

    34

    28

    38

    37

    38

    34

    32

    38

    35

    40

    40

    41

    42

    41

    RhoBTB2

    70

     

    32

    32

    31

    29

    34

    28

    39

    37

    38

    33

    32

    39

    37

    40

    40

    42

    42

    41

    RhoH

    34

    32

     

    29

    32

    36

    38

    33

    40

    40

    41

    41

    38

    40

    39

    42

    40

    40

    41

    40

    Rnd1

    32

    32

    29

     

    53

    61

    37

    39

    41

    42

    41

    31

    32

    36

    34

    37

    37

    39

    39

    38

    Rnd2

    31

    31

    32

    53

     

    63

    39

    41

    46

    47

    43

    28

    31

    36

    35

    37

    41

    40

    41

    39

    Rnd3

    29

    29

    36

    61

    63

     

    37

    40

    48

    48

    47

    31

    32

    39

    35

    38

    41

    39

    42

    40

    RhoD

    34

    35

    38

    37

    39

    37

     

    49

    49

    49

    49

    39

    36

    42

    38

    43

    44

    46

    49

    49

    RhoF

    28

    28

    33

    39

    41

    40

    49

     

    47

    48

    47

    36

    37

    46

    43

    43

    46

    50

    59

    47

    RhoA

    38

    39

    40

    41

    46

    48

    49

    47

     

    92

    85

    40

    44

    51

    48

    53

    55

    53

    57

    55

    RhoC

    37

    37

    40

    42

    47

    48

    49

    48

    92

     

    85

    40

    44

    50

    49

    51

    55

    53

    57

    54

    RhoB

    38

    38

    41

    41

    43

    47

    49

    47

    85

    85

     

    42

    45

    51

    48

    50

    53

    54

    55

    54

    Wrch2

    34

    33

    41

    31

    28

    31

    39

    36

    40

    40

    42

     

    59

    51

    48

    53

    46

    51

    52

    53

    Wrch1

    32

    32

    37

    32

    31

    32

    36

    37

    44

    44

    45

    59

     

    50

    46

    56

    48

    54

    54

    54

    TC10

    38

    39

    40

    36

    36

    39

    42

    46

    51

    50

    51

    51

    50

     

    76

    66

    54

    60

    62

    61

    TCL

    35

    37

    39

    34

    35

    35

    38

    44

    48

    49

    48

    48

    46

    76

     

    63

    53

    58

    60

    59

    Cdc42

    40

    40

    42

    37

    37

    38

    43

    43

    53

    51

    50

    53

    56

    66

    63

     

    61

    69

    71

    70

    RhoG

    39

    40

    40

    37

    41

    41

    44

    46

    55

    55

    53

    46

    48

    54

    53

    60

     

    72

    72

    70

    Rac2

    41

    42

    40

    39

    40

    39

    46

    50

    53

    53

    54

    50

    54

    60

    58

    69

    72

     

    92

    89

    Rac1

    42

    42

    41

    39

    41

    42

    49

    49

    57

    57

    55

    52

    54

    62

    60

    71

    72

    92

     

    93

    Rac3

    41

    41

    40

    38

    39

    40

    49

    47

    55

    54

    54

    53

    54

    61

    59

    70

    70

    89

    93

     
    Compared with other Ras superfamily proteins, the Rho-family small G proteins have a Rho insert region, which is a unique α-helical sequence located between the fifth β strand and the fourth α helix in the G domain [5]. Many of the typical Rho-family GTPases only have a Rho-type G domain with short N- and C-terminal extensions [6][7]. Similar to other Ras superfamily members, they also have a hypervariable region encoding a prenylation site at their C-termini and sometimes a polybasic region. These signals allow them to associate with specific membrane compartments [8].
    Additionally, some of them also possess a CAAX (C: Cys, A: aliphatic residue, X: any amino acid) tetrapeptide sequence at their C-termini, which is important for lipid modification. This process involves three main steps, which are isoprenylation, proteolysis and carboxyl methylation [9]. An isoprenoid lipid is attached to the CAAX box by a prenyltransferase, such as geranylgeranyltransferase (GGTase) or farnesyltransferase (FTase). Both of these enzymes recognise the CAAX sequence before adding a 20-carbon geranylgeranyl or 15-carbon farnesyl to the cysteine residue of the CAAX sequence via a thioether linkage. Prenylation is followed by the proteolysis of the three C-terminal residues (AAX) by a prenyl protein peptidase (from the Rce1 family as an example) to release AAX. The prenylated cysteine is then methylated by isoprenyl-cysteine carboxyl methyl-transferase. Compared with the atypical small RhoGTPases, many of the typical Rho-family GTPases are geranyl-geranylated or farnesylated, and only a few, such as RhoB, TC10 and Rac1, are palmitoylated (Table 2) [8][10]. However, some of the atypical small Rho GTPases also have a functional CAAX box that allows them to undergo prenylation (Table 2). For instance, both RhoU and RhoV only undergo palmitoylation, while Rnd proteins undergo farnesylation. Conversely, the RhoBTB subfamily does not possess any canonical CAAX motifs, suggesting that they may not undergo any lipid modification at the C-terminal.
    Table 2. C-terminal sequences of the Rho-family small GTPases and their lipid modification.

    Group

    Rho Protein

    C-Terminal Sequence

    Lipid Modification

    Ref

    Typical

    RhoA

    KDGVREVFEMATRAALQARRGKKKSGCLVL

    GG

    [11]

    RhoB

    VREVFETATRAALQKRYGSQNGCINCCKVL

    GG, F, P

    RhoC

    KEGVREVFEMATRAGLQVRKNKRRRGCPIL

    GG

    Rac1

    RGLKTVFDEAIRAVLCPPPVKKRKRKCLLL

    GG, P

    Rac2

    RGLKTVFDEAIRAVLCPQPTRQQKRACSLL

    GG

    Rac3

    RGLKTVFDEAIRAVLCPPPVKKPGKKCTVF

    GG

    RhoG

    QDGVKEVFAEAVRAVLNPTPIKRGRSCILL

    GG

    Cdc42

    QKGLKNVFDEAILAALEPPEPKKSRRCVLL

    GG

    TCL

    AVFDEAILTIFHPKKKKKRCSEGHSCCSII

    F

    TC10

    DEAIIAILTPKKHTVKKRIGSRCINCCLIT

    F, P

    Atypical

    RhoU

    QQQPKKSKSRTPDKMKNLSKSWWKKYCCFV

    P

    [11]

    RhoV

    EHKARLEKKLNAKGVRTLSRCRWKKFFCFV

    P

    RhoD

    AVFQEAAEVALSSRGRNFWRRITQGFCVVT

    F, GG

    [12]

    RhoF

    EDVFREAAKVALSALKKAQRQKKRRLCLLL

    F, GG

    Rnd1/ RhoS

    LSKRLLHLPSRSELISSTFKKEKAKSCSIM

    F

    [11]

    Rnd2/ RhoN

    MQRSAQLSGRPDRGNEGEIHKDRAKSCNLM

    F

    Rnd3/ RhoE

    KRISHMPSRPELSAVATDLRKDKAKSCTVM

    F

    RhoH/ TTF

    VFECAVRTAVNQARRRNRRRLFSINECKIF

    GG, F

    [11][12]

    RhoBTB1

    KREREKEDIALNKHRSRRKWCFWNSSPAVA

    Unknown

    N/A

    RhoBTB2

    KRRWLFWNSPSSPSSSAASSSSPSSSSAVV

    Unknown

    Putative palmitoylated cysteines are in boldface type and coloured red and CAAX prenylation motifs are underlined. N/A: not applicable.
    Among the Rho-family GTPases, the most extensively studied members are RhoA, Rac1 and Cdc42. These proteins and their subfamilies are known as classical or typical Rho-family GTPases. The other Rho-family proteins are known as the non-classical or atypical Rho GTPases and include the RhoBTB and Rnd subfamilies. This classification is made based on the ability of the Rho-family GTPases to undergo the standard GTPase cycle [6][13]. The atypical Rho GTPases often contain extra domains and are therefore longer than classical Rho GTPases.
    Compared with the atypical small GTPases are further divided into fast-cycling and GTPase-defective G proteins (Table 3). In resting cells, the typical Rho-family GTPases are generally assumed to be in their inactive GDP-bound form either alone or in complex with RhoGDIs [14]. However, in contrast to the typical Rho-family GTPases, RhoD, RhoF, RhoU and RhoV have a rapid nucleotide exchange compared with standard GTP hydrolysis, which classifies them as fast-cycling small G proteins [15][16][17]. The Phe28 mutations of Ras, Rac1, Cdc42 and RhoA have rapid nucleotide exchange and can induce oncogenic transformation [18][19]. RhoU and RhoV also have mutated phenylalanine at position 28 (Table 3), and this factor might be one of the reasons for considering both of these proteins as fast-cycling small G proteins.
    Table 3. Selected amino acid sequences of the typical and atypical small Rho GTPases.

    Amino Acids 12, 59 and 61

    Group

    Subfamily

    Member

    Sequence

    Classic

    Cdc42

    Cdc42

    12        59    61

    Cells 10 00950 i001         Cells 10 00950 i002      Cells 10 00950 i003

    GDGAV---AGQED

    Fast-cycling

    RhoU/RhoV

    RhoU

    GDGAV---AGQED

    RhoV

    GDGAV---AGQDE

    RhoD/RhoF

    RhoD

    GDGGC---AGQDD

    RhoF

    GDGGC---AGQED

    GTPase defective

    RhoBTB

    RhoBTB−1

    GDNAV---FGDHH

    RhoBTB−2

    GDNAV---FGDHH

    Rnd

    Rnd1

    GDVQC---SGSPY

    Rnd2

    GDAEC---SGSSY

    Rnd3

    GDVQC---SGSPY

    RhoH

    GDSAV---AGNDA

    Amino acids 28

    Classic

    Rac

    Rac1

    28

    Cells 10 00950 i004

    SYTTNAFPGEYIP

    Fast-cycling

    RhoU/RhoV

    RhoU

    SYTTNGYPTEYIP

    RhoV

    SYTCNGYPARYRP

    RhoD/RhoF

    RhoD

    VFADGAFPESYTP

    RhoF

    VYSQGSFPEHYAP

    Amino acid residues 12, 28, 59 and 61 are in boldface type and underlined [13].
    The fast-cycling Rho GTPases have GTPase activity and are identical to Cdc42 at amino acids Gly12, Ala59 and Gln61 (Ras numbering) (Table 3) [13]. Gly12 and Ala59 are important for GTP hydrolysis, while Gln61 is necessary to stabilise the transition state of hydrolysis [20]. These amino acid residues are reported to be mutated in GTPase-deficient Ras [21]. By contrast, the GTPase defective atypical Rho-family members, including Rnd1, Rnd2, Rnd3, RhoBTB1, RhoBTB2 and RhoH, do not possess the conserved Gly12, Ala59 and Gln61 compared with Cdc42 (Table 3) [4][13].
    Most studies have focused on the role of the typical small Rho GTPases in diseases, but information on the atypical small Rho GTPases is limited. Among the rare small Rho GTPases that receive much attention is RhoH as it is associated with several diseases, such as B cell malignancies, immunodeficiency and autoimmune diseases. This small GTPase has a unique role compared with others because it acts as a tumour-suppressor protein. Hence, a thorough understanding of the function of RhoH in both normal and disease conditions may help to identify and design an efficacious target.

    2. RhoH, An Atypical Rho Family Small GTPase

    Within the family, RhoH and Cdc42 share 42% sequence similarity, with the major differences in the C-terminal region (Figure 1). It also contains a shorter Rho insert region that is more similar to classical Rho-family proteins but is GTPase defective because conserved residues corresponding to Gly12, Ala59 and Gln61 (Ras numbering) are absent (Table 3). However, RhoH has a CAAX box, a CKIF motif at its C-terminus, which allows farnesylation and geranyl-geranylation [22] and facilitates membrane targeting. RhoH myristoylation at the N-terminal may also assist RhoH attachment to the plasma membrane and promote its interacting partners, Lck, ZAP−70 and Syk recruitment to the membrane to facilitate T cell receptor (TCR) signalling [23][24][25]. Among the Rho family members, RhoH is the only member with an ITAM-like motif, which is characterised by two tyrosines, spaced by six amino acids: Y73xxA766Y83xxA86 (Figure 1). This consensus motif is crucial for the recruitment of ZAP−70 to CD3ζ in the immunological synapse [23].
    Figure 1. Sequence alignment and architecture of RhoH. Sequence alignment of RhoH, Rnd2, Wrch2, RhoA, Rac1 and Cdc42. Rnd2 and Wrch2 are representatives of the fast-cycling and GTPase-defective small GTPases, while RhoA, Rac1 and Cdc42 are typical small Rho GTPases (Top). RhoH shares a phosphate-binding loop with other small Rho GTPases but is assumed as GTPase defective due to the absence of Gly12. The Rho-specific insert in RhoH is shorter than that in other small Rho GTPases. RhoH also has an ITAM-like motif at position Tyr73 to Ala86 and a carboxyl-terminal insert domain (L182FSINE187) that function to regulate RhoH stability. RhoH also undergoes prenylation at the C-terminal due to the presence of the CAAX box (CKIF), similar to other typical small Rho GTPases (Bottom). (Created with Biorender).
    Given that RhoH may be GTPase defective, other mechanisms have been described to regulate RhoH activity, including the regulation of mRNA level and tyrosine phosphorylation of the unique ITAM-like motif [22]. Additionally, RhoH activity can be regulated by lysosomal degradation through its unique C-terminal region, L182FSINE187 domain, located in between its polybasic region and CAAX box. This insert domain has been shown to regulate RhoH stability via chaperone-mediated autophagy (CMA) [26].
    In contrast to the typical Rho-family members, which are best studied for their role in promoting actin cytoskeleton reorganisation, especially during cell division and migration [2], RhoH does not regulate actin reorganisation in NIH3T3 or MDCK cells [27]. However, Mino et al., (2018) showed that RhoH is involved in modulating the structure of actin-cytoskeleton and transcriptional activity during T cell migration and adhesion by forming a multi-protein complex with p120 catenin and the transcriptional regulator, Kaiso, to attenuate Rac1 signalling. Aside from regulating Rac1 signalling, RhoH interaction with Kaiso is necessary to facilitate Kaiso nuclear localisation to repress BCL6 gene, a transcriptional repressor expression, resulting in an increase in tumour suppressor p53 protein [28]. Collectively, the inhibitory function of RhoH would help to reduce the events of cell survival, migration and invasion [29][30]. By contrast, although RhoH is assumed to be expressed only in haematopoietic cells, it was shown to promote cell migratory polarity in prostate cancer cell line by directing Rac1 and PAK2 to membrane protrusions [31]. These contrary functions of RhoH might be dependent on the type and origin of the cells. For instance, RhoH is dispensable for the development of myeloid, erythroid, and B cells but is crucial for T cell production, survival and migration [32][33]. This function is consistent with the study of Chae et al., (2010) that showed a lack of RhoH-impaired thymocyte development and stimulation of peripheral T cell unresponsiveness [23][34].
    The current knowledge of RhoH function is only limited in T cells (Figure 2), whereupon during response to TCR activation, RhoH is phosphorylated by kinases, such as DRAK2 [35]. This phosphorylation event promotes its interaction with ZAP−70 via the ITAM-like motif. ZAP−70 and Lck will then be recruited to the TCR, where ZAP−70 is activated and colocalised with its substrates. As a result, activated ZAP−70 will promote linker of activated T cells (LAT) and SLP76 signalling in T cell, crucial for T cell activation and development [23][24][25][36]. Additionally, RhoH was also found to act as an adaptor protein that retains Lck in an inactive state, thereby suggesting that RhoH can regulate both pre-TCR and TCR signalling during T cell development. However, in response to ligand-mediated TCR activation, Lck is recruited to the membrane by RhoH and dephosphorylated by CD45, resulting in Lck auto-activation and its release from RhoH [37]. Active Lck will then stimulate PI3K signalling and the activity of RhoGEF, Vav1. Nonetheless, despite the obvious role of RhoH in T cell development, RhoH involvement in T cell-specific malignancies, such as in T cell lymphomas, is not well understood. Conversely, RhoH is known to be associated with B-cell neoplasm, but its function in regulating B cell signalling should be further studied.
    Figure 2. RhoH function upon TCR activation. The illustration shows the role of RhoH in regulating TCR signalling. Upon TCR stimulation, RhoH is phosphorylated by DRAK2 that promotes the interaction between RhoH and ZAP−70 via the ITAM-like motif of RhoH. ZAP−70 is recruited to the TCR CD3ζ chain, thereby activating LAT and SLP76 signalling. As for Lck, RhoH functions to assist Lck recruitment to the membrane to facilitate TCR signalling. Active Lck promotes PI3K signalling and PIP3-induced DOCK8 activation. As a result, Rac1-related signalling is activated. Vav1-induced Cdc42 activation can also be regulated by Lck. However, at rest, RhoH recruits Lck to the plasma membrane, where RhoH accelerates and supports the inhibitory phosphorylation of Lck at Y505 (labelled with P5) by CSK (Created with Biorender).

    3. RhoH as a Therapeutic Target

    Considering the importance of RhoH in regulating TCR and BCR signalling pathways, much effort has been exerted on targeting RhoH to control diseases derived from abnormal T and B cell activation, such as cancer, immunodeficiency disorders and autoimmune diseases. RhoH protein levels are frequently reduced in the aforementioned diseases, suggesting the need to rectify the aberrant RhoH expression in the haematopoietic cells. The current drugs known to modulate RhoH activities are lenalidomide and ibrutinib [38]. However, the use of these drugs is critically dependent on the types of disease as these drug treatments were shown to regulate RhoH protein expression. For instance, the use of lenalidomide in the CLL murine model results in a decreased RhoH protein expression that promotes Rac1 and RhoA activation [39]. Additionally, deregulated RhoH protein expression can be corrected by using the current approach that relies on the potential therapeutic opportunity to target CMA [40]. This factor is based on the understanding that cellular protein levels are significantly influenced by protein stability. Hence, regulating their modes of degradation may promote protein stability. For instance, all-trans retinoic acid can be used to block the inhibitory effect of retinoic acid receptor alpha (RARα) on the CMA process, especially on LAMP2A expression and its trafficking to lysosomes [41]. This approach can be applied to RhoH as the presence of the inserted domain, LFSINE, at the C-terminal was shown to act as a recognition signal for lysosomal uptake and CMA-mediated degradation, while not affecting its function on both T cells and B cells [26].
    Another alternative is by targeting the RhoH interacting partner, ZAP−70. RhoH mediates TCR-induced ZAP−70 activation by recruiting it to CD3ζ in the immunological synapse [23][32][34]. Additionally, RhoH was also found to promote ZAP−70-induced BCR signalling, which has been implicated in CLL [33][42][43][44]. Hence, preventing ZAP−70 recruitment to the plasma membrane may help to block excessive signalling. Several studies have identified potential ZAP−70 inhibitors that can modulate the interaction with TCR [45]. However, despite its therapeutic potential in autoimmune diseases and organ transplant rejection in vitro and in vivo, these inhibitors have never been tested in clinical trials, indicating the need for further investigation.
    Alternatively, a peptide that can block or stimulate RhoH-related signalling may be used as a potential therapeutic target. The use of therapeutic peptides is an emerging targeted therapy that is often associated with rapid production and capacity for modification. However, this strategy has several drawbacks that usually arise due to their limited bioavailability and specificity [46][47]. Thus, far, no specific RhoH-therapeutic peptide is available or has been reported that may be due to the limited knowledge of RhoH functional activities.

    The entry is from 10.3390/cells10040950

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