3.1. Differentiation and Development
In multiple animal models, various complex subunits have been ascribed functions in developmental and cell differentiation pathways. In mice, RanBP9 knockout resulted in both sexes being sterile due to defects in oogenesis and spermatogenesis
[57][58]. In
D. melanogaster, two groups exhibited fascinating function and regulation of the entire complex as part of the precise temporal control of the maternal proteome in the maternal-to-zygotic transition (MZT). In the early stages of the MZT, the
D. melanogaster CTLH complex is activated by translational upregulation of the UBE2H homologue, causing the CTLH-dependent degradation of RNA-binding components of a translation-inhibiting complex required for oogenesis
[45][52].
An important role for complex members in red blood cell homeostasis has been well documented. An initial study showed that
Maea−/− mouse embryos died perinatally with anemia and differentiation defects in erythroid and macrophage lineages, primarily caused by defective erythroblast enucleation
[59]. WDR26 has also been associated with regulating red blood cell development.
Wdr26 expression is upregulated in terminally differentiating erythroblasts and its knockdown caused severe defects in enucleation, a reduction in hemoglobin production, and blocked differentiation at the basophilic erythroblast stage
[46]. Furthermore,
Wdr26−/− zebrafish exhibited profound anemia likely due to defective erythropoiesis, a phenotype also reported in the initial study on
Maea−/− mouse embryos
[46][59]; however, rather than observing defects in erythroblastic island adhesion or macrophage differentiation, the Wdr26 knockout animals had deficiencies in the nuclear opening of erythroblasts. This led to the discovery that the CTLH complex directs the polyubiquitination and degradation of lamin B, which facilitates enucleation
[46].
Overall, there is a clear importance of CTLH complex subunits in different aspects of development and differentiation. Thus far, however, the ubiquitination activity has only been linked to the degradation of RNA binding proteins during MZT in
D. melanogaster and degradation of lamin B for nuclear condensation in differentiating mammalian erythroblasts
[45][46][52]. More mechanisms and ubiquitin targets in developmental contexts are likely to be revealed soon.
3.2. Cell Migration and Adhesion
The mammalian complex has been associated with several cell migration and adhesion pathways. Reports have shown RanBP9 association with various integrin, junctional, receptor, and adhesion proteins (reviewed in
[29]). The depletion of RanBP9 increased HT22 and NIH3T3 cell attachment by disrupting focal adhesion signaling
[60] and breast cancer cell invasiveness by regulating BLT2-mediated reactive oxygen species generation and IL-8 production
[61]. Muskelin was initially identified in a screen for proteins that promoted C2C12 mouse myoblast cell line adherence to a thrombospondin-1 substratum
[62]. In rat lens epithelial cells, muskelin depletion reduced Rho-GTP activation, myosin phosphorylation, the dissociation of stress fibers, and cell migration
[63]. Muskelin and RanBP9 depletion in lung A549 cells adherent on fibronectin caused enlarged cell perimeters and altered morphology and F-actin distribution
[64]. WDR26 has been linked with cell migration in multiple cell types, but with opposing effects observed. In leukocytes, WDR26 is required for SDF1α-induced cell migration and promotes PI3K/Akt-signaling-mediated migration and invasiveness in MDA-MB-231 breast cancer cells
[65]. In intestinal epithelial cells, however, WDR26 was found to inhibit FPR1-mediated cell migration and wound healing
[66].
Thus far, the only direct implication involving the entire complex in cell migration is through a negative regulation of histone deacetylase 6 (HDAC6) activity, which is likely responsible for the increased cell migration observed in RanBP9-deficient HEK293 cells
[67]. Cells depleted of RanBP9, muskelin, and RMND5A showed increased HDAC6 activity and/or increased deacetylation of HDAC6 target α-tubulin, but no change in HDAC6 protein levels, whereas RanBP9, MAEA, and GID8/TWA1 were shown to be colocalized at microtubules with HDAC6
[67]; however, in this context, ubiquitination was not investigated, so the regulatory mechanism of HDAC6 by the CTLH complex remains unclear. The ubiquitination of HDAC6 that alters its activity or the ubiquitination of an HDAC6 coregulator are two possible mechanisms underlying HDAC6 regulation by the CTLH complex.
3.3. Nuclear Functions
The CTLH complex is implicated in the nuclear condensation of developing erythroblasts via direct polyubiquitination of lamin B
[46]. Beyond this, an exact nuclear role is unclear, but chromatin regulation is likely because at least two complex members have been found together in the interactomes of several critical transcription factors or DNA repair proteins
[68][69][70][71][72][73]. Furthermore, UBE2H has been linked to histone ubiquitination
[74][75][76], but whether this involves the CTLH complex is unknown.
In support of a role in transcription, microarray analyses of RanBP9 Hela and HCT116 knockdown cells indicated numerous effects on gene expression
[77]. RanBP9 and/or RanBP10 interactions with steroid and hormone nuclear receptors, such as the androgen receptor and glucocorticoid receptor, have been observed, and both have been shown to act as transcriptional co-activators for these proteins
[78][79][80]. RanBP9 also interacted and enhanced transcriptional activities of Epstein–Barr virus (EBV) proteins Rta and Zta, and was present on Zta-responsive elements on EBV gene promoters
[81][82]. Sumoylation of the viral transcription factors by Ubc9 was regulated by RanBP9, which affected their transcriptional activity
[81][82]. Thus far, that is the only established direct mechanism for any complex member on transcriptional regulation.
3.4. Cell Proliferation, Death, and Survival Pathways
Despite the overall conservation between the yeast and mammalian complexes, the mammalian (human or mouse) complex does not regulate gluconeogenesis, and does not ubiquitinate human Fbp1, likely because, as already mentioned, the degrons are not the same
[14][83][84]. Instead, the human complex has been demonstrated to inhibit the opposite pathway, glycolysis, by regulating the ubiquitination of enzymes PKM and LDHA
[48]. Instead of degradation, however, PKM and LDHA activities were increased in RanBP9-deficient cells, and global proteomic and ubiquitinome analyses suggested that non-degradative ubiquitination by the complex may be prevalent
[48]. A corresponding increased glycolytic flux and altered metabolism was observed in RanBP9-deficient HeLa cells
[48], a hallmark of cancer cells which enables them to survive as highly proliferating cells
[85].
Several connections of the complex with the WNT pathway have been established. A recent report claimed that RMND5A-MAEA can directly ubiquitinate β-catenin; however, no in vitro ubiquitin assay or binding assay was conducted
[86]. The same group previously published that WDR26 associated with Axin, but not with β-catenin
[53]. The depletion of WDR26 increased β-catenin stability in
X. laevis and in WNT-stimulated HEK293 cells independently of GSK3β, and regulated β-catenin ubiquitination if co-expressed with Axin. Interestingly, the entire complex was found in the Axin interactome
[87], and MAEA and WDR26 were present in the APC interactome with decreased binding after WNT stimulation
[88]. In
D. melanogaster, β-catenin accumulates in RanBP9 null terminal filament cells of the germ stem cell niche
[89].
Some complex members have been associated with the activation of apoptosis in response to cellular stress. In response to IR, RanBP9 has been reported to be phosphorylated in an ATM-dependent manner and initially predominantly nuclear immediately after IR treatment, but then increasingly cytoplasmic as treatment is prolonged
[90][91]. At 72 h of IR treatment, RanBP9 is recruited to perinuclear aggresomes
[92]. Studies in lung cancer cells showed that RanBP9 is essential for DNA damage response activation, homologous recombination DNA repair, and sensitivity to genotoxic stressors such as IR and cisplatin treatment
[90][93]. In
Ranbp9 germ cell knockout testes, enhanced apoptosis of spermatocytes and defective DNA repair is also observed
[58]. On the other hand, RanBP9 has been shown to be pro-apoptotic in a variety of cell lines via activation of the intrinsic pathway, as well as through other means, such as regulation of the MAPK pathway, aggresome formation, the activation of cofilin, and interactions with p73 and TSSC3
[91][92][94][95][96][97]. In keratinocytes, ARMC8 expression had a subtle positive effect on apoptosis induction in response to ultraviolet B radiation
[98]. Meanwhile, WDR26 expression inhibited oxidative-stress-induced cell death in SH-SY5Y cells and cardiomyocytes
[99][100].
3.5. Functions and Disease Implications in the Central Nervous System
Beyond roles in the development of the brain, a few complex subunits have been linked to neuron signaling and neurodegenerative diseases. For example, in the mouse brain, muskelin is required for normal hippocampal network oscillation and for controlling lysosomal degradation of the cellular prion protein (PrPC) and GABA
A receptor (GABA
AR)
[101][102]. Both muskelin and RanBP9 have separately been shown to associate with amyloid precursor protein (APP)
[102][103]. In RanBP9-overexpressing mice, APP processing and Aβ generation is elevated, resulting in the increased deposition of amyloid plaques (a hallmark of AD)
[60][103][104]. RanBP9 overexpression may also contribute to AD progression by stabilizing Tau protein through interaction with Hsp90/Hsc70
[105]. Although no other complex member has been functionally or genetically linked to AD pathogenesis, UBE2H mRNA is significantly higher in the blood of AD patients
[106]. It remains unclear if there are functional relationships between RanBP9, muskelin, and other complex members in the adult brain.
3.6. Immune System
There are some reports of roles of CTLH complex members in immunology, although nothing linking the entire complex. The UBE2H promoter contains an NF-κB binding site and is upregulated by the proinflammatory cytokine tumor necrosis factor α (TNF-α) as part of an overall increased ubiquitin conjugating activity observed upon TNF-α treatment
[107]. A compelling study discovered that RanBP9 is part of a complex with AXL and LRP-1 that facilitates dendritic cell efferocytosis and antigen cross-presentation to T cells
[108]. Additionally, RanBP9 was shown to interact with TRAF6 and suppress the TRAF6 activation of NF-κB signaling
[109]. In
D. melanogaster, the RanBP9/10 homologue was identified as a negative regulator of the cytokine-activated Janus kinase (JAK)/signal transducer and activator of the transcription (STAT) pathway
[110].
A connection with viruses was suggested by the presence of the CTLH complex subunits in the interactomes of viral proteins from severe acute respiratory syndrome coronavirus 1
[111][112], Kaposi’s sarcoma-associated herpesvirus
[113], and β-herpesvirus human cytomegalovirus
[114]. Functionally, RanBP9 and RanBP10 have been identified as host proteins required for viral replication
[81][82][115]. Overall, these studies provide evidence that the CTLH complex is involved in immune and viral regulations, although the ubiquitin activity has not yet been implicated.
3.7. Endocytosis
Some complex members have been implicated in the internalization of various proteins and endocytosis/lysosomal pathways, an intriguing connection to yeast GID complex regulation of Fbp1 degradation in the vacuole. RanBP9 modulates APP, LRP, and β1-integrin endocytosis in neurons
[60][103]. ARMC8 has been shown to promote the interaction of the endosomal sorting complex required for transport (ESCRT) complex with ubiquitinated proteins
[116]. As mentioned, muskelin promotes the internalization and degradation of GABA
AR in mouse neurons
[101]. Muskelin interacts with GABA
AR at the plasma membrane rich in F-actin, where the two proteins associate with Myosin VI. There, muskelin bridges associations of GABA
AR with dynein and promotes transport in a multivesicular body and subsequent degradation in the lysosome, instead of recycling back to the membrane. It is a process quite reminiscent of the yeast GID-complex-mediated internalization of Fbp1 and subsequent delivery to the vacuole (which, of course, does not involve muskelin) and awaits further investigation to confirm whether other CTLH members are involved in this internalization and transport mechanism.