These enzymes exert two crucial functions: they determine the specific type of ubiquitinated substrate and permit the final transfer of Ub (to the substrate) [34]. In this report, E3 Ub ligases were grouped into 3 families according to [35]: the HECT (homologous to the E6-AP carboxyl terminus), RBR (RING-in-between-RING), and RING (really interesting new gene) E3 families.
3.1.5.2 RING E3 Ub ligases
RING E3 constitute the largest family of E3 Ub ligases [8] [34]. For instance, analysis of the mouse genome identified 398 putative E3 [46]. In the following sections, RING E3 Ub ligases genes were classified into three main subgroups: i) single subunits, ii) multi-subunits RING E3 and iii) U-Box RING E3.
A. Single subunits RING E3
The list established by [47] was used for the analysis of the major E3 subgroups : Cbl, Deltex, Goliath, IAP, Listerin, Makorin, MARCH, Neuralized, Pellino, Pex, Polycomb, Praja, RBR, Siah, Traf, Trim and Ubr. A large heterogeneity in expression levels was observed among these genes. The most expressed subgroup includes: Deltex, Goliath, Makorin, March, Neuralized, Praja, Polycomb, Traf (Fig. 4) and Trim (Fig. 5). The minor expression of other genes (Cbl, IAP, Listerin, Pellino, Pex, Siah, and Ubr) is analyzed in Table 1. They all displayed TPM values < 37, except Ubr7 having TPM values of ⁓60 at E11 and E13.
Table 1 It gives the list of the genes coding for RING E3 ligases that were found to be weakly expressed during the formation of the cerebral cortex. They all displayed TPM values <65.
n.d.: not detected, below the detection threshold. Depending on the gene, the abundance of transcripts increased, decreased or was nearly constant (ä, æ and =, respectively) during corticogenesis.
Deltex E3 Ub ligases
Dtx3 and Dtx4 were the major deltex genes with TPM values increasing from 179 to 351 (Dtx3) and from 40 to 91 (Dtx4) (Fig. 4A), revealing a strong positive regulation during corticogenesis. However, the most regulated gene of this group was Dtx1: no transcripts were detected at E11 but a strong induction was noted afterwards with TPM values increasing from 9 (at E13) to 60 (at PN1), a nearly 7-fold increase (Fig. 4A). Deltex E3 has been principally studied in the context of tumorigenesis and invasion [48] but very little is known regarding the roles played by these Deltex E3 Ub ligases in the developing or adult brain in mammals. The marked enhancement of the deltex genes expression supports key roles in neuronal growth and differentiation in mammals.
Goliath E3 Ub ligases
Twenty-nine ortholog genes of the Drosophila Goliath E3 Ub ligases were identified in mouse (https://flybase.org/reports/FBgg0000104.html). Nine of them were not expressed (Rnf43, Rnf128 (Grail), Rnf133, Rnf148, Rnf150, Znrf3, Znrf4, Zswim2 and 4930595M18Rik). In this subgroup, Rnf44 (68–115 TPM), Rnf167 (72–125 TPM), and Rnf126 (46–70 TPM) were the genes mostly expressed (Fig. 4B). Of note, Rnf215 was the most regulated gene with an abundance of transcripts decreasing from 74 to 27 TPM from E11 to PN1, a 2.7 fold decrease during corticogenesis. Nothing is known concerning the contribution of RNF44 in brain formation and functions. The E3 ligase RNF167 plays important roles in neuronal cells. Though principally found in lysosomes, a fraction of RNF167 is present at the cell surface where it participates in the ubiquitination of the AMPA receptors. This process modulates the number of AMPA receptors at the cell surface as well as synaptic currents. Therefore, RNF167 is an important physiological modulator of glutamatergic neurotransmission [49]. This RNF167-dependent ubiquitination of AMPA receptors was recently shown to occur under the control of E2 enzymes Ube2D1 and Ube2N [50]. RNF126, another prominent actor of this subgroup, has been shown to be involved in Friedreich ataxia, a severe genetic neurodegenerative disease characterized by a reduced expression of the essential mitochondrial protein frataxin. The E3 Ub ligase RNF126 specifically mediates frataxin ubiquitination, which induces its degradation [51]. Our results points towards a role in neuronal function from early embryonic stages.
Makorin E3 Ub ligases
All three makorin genes (Mkrn1-3) were expressed in the immature cerebral cortex (Fig. 4A). Mkrn1 was the predominant gene with TPM values augmenting from 32 (at E11) to 116 (at E17). Consistent with our findings, Mkrn1 was originally identified as a highly expressed gene during mouse embryonic development with a high level of mRNA expression in the developing brain [52]. It was recently reported low brain levels of makorin 1 proteins despite a relative high mRNA abundance due to the auto-ubiquitination properties of this E3 Ub ligase inducing its proteasomal degradation [53].. Experiments performed on Xenopus embryos showed that Mkrn2 inhibits neurogenesis acting downstream of phosphatidylinositol 3-kinase (PI3K) and Akt [54]. Thus, Mkrn proteins certainly play major functions in the developing nervous system.
MARCH E3 Ub ligases
The family of proteins of the membrane-associated RING-CH (MARCH) comprises eleven E3 Ub ligases (MARCH-1 to 11) [55]. Four March genes out of the eleven analysed were not expressed: March-1, -3, -10 and − 11. The major gene of the group was March-9. Its TPM values were around 60 at E13 but strongly augmented at E17 (192 TPM) and PN1 (217 TPM), corresponding to a > 3.5-fold augmentation (Fig. 4A). At E17 and PN1, March-9 transcripts represented > 50% of all March transcripts. In dendritic cells, MARCH-9, localizes in the trans-Golgi network (TGN) and controls a TGN-to‐endosome transport step [56]. MARCH-9 expression has been mainly found in immune cells and organs like lung, lymph node, and spleen rather than neuronal cells [57]. Our data however indicate that MARCH-9 was highly expressed at the end of neurogenesis. Clearly, additional work is needed to delineate the neuronal functions of MARCH-9 proteins in the brain. Though much less expressed, the other major March gene was March-5, a mitochondrial-associated E3 Ub ligases. It had TPM values of 58–62, with no sign of development regulation. Of note, transcripts of March-4 were detected at E17 and onward with TPM values of ⁓12 (Fig. 4A). MARCH-4, a Golgi-associated E3 Ub ligase, was the only member of the MARCH family previously known to be expressed in the brain [57] while the TPM values were extremely limited (below 13 TPM) (Fig. 4A). Our transcriptomic analysis revealed a large repertoire of actors, with seven March genes expressed throughout corticogenesis and both a preponderant and highly regulated expression of March-9 (Fig. 4A).
Neuralized E3 Ub ligases
Transcripts of three neuralized genes were measured: Neurl1b, Neurl2 and Neurl4. The expression of the major gene Neurl4 was enhanced with TPM values augmenting from ⁓90 to ⁓200 (Fig. 4A). The protein NEURL4, expressed in the developing rodent cerebellum [58], is a p53 interacting protein that inhibits cellular growth when overexpressed [59]. Furthermore, experiments conducted on NEURL4 knockdown animals showed a reduced number of presynaptic boutons, indicating that NEURL4 regulates synapse development in the brain [58] consistent with its up-reglated expression during corticogenesis.
Praja E3 Ub ligases
The expression of the two Praja genes Pja1 and Pja2 was positively regulated during corticogenesis. The abundance of Pja1 and Pja2 transcripts increased by a factor 1.6 and 3.8, respectively (Fig. 4A). Although less regulated, the Pja1 gene was by far the most predominant Praja gene and one of the most expressed gene of the Ub system. Its TPM values were in the order of 270 (at E11) and ⁓430 (at PN1) with a peak at E17 (⁓480) (Fig. 4A). On average, Pja1 transcripts were 11 and 5–6 times more abundant than Pja2 transcripts at E11-E13 and E17-PN1, respectively. The human and mouse Pja1 genes are highly expressed in the brain, particularly in the cerebral cortex [60]. Northern blots experiments detected Pja1 mRNA in the immature brain at E11.5. Suppression of Pja1 expression leads to high levels of apoptosis, indicating that this protein could exert a pro-survival anti-apoptotic role. In line with a function in cell survival, Pja1 mRNA is overexpressed in twenty-nine cancer types, particularly in gliomas [60]. Altogether, these results support an important role for Praja1 proteins in brain development and regulation of cell apoptosis.
Polycomb complexes
Polycomb containing complexes possess an E3 Ub ligase activity due to RING1A (Ring1) or RING1B (Rnf2). The abundance of Rnf2 transcripts did not vary during corticogenesis whereas a reduction of Ring1 transcripts was observed between E11 and E17 with TPM values diminishing from 80 to 50 (Fig. 4A).
Traf E3 Ub ligases
Transcripts of four Traf genes were found but at various levels, with the predominant Traf4 having TPM values of 120–180 (Fig. 4A). In contrast to the other Traf members, its expression was enhanced during corticogenesis. TRAF4 proteins are essential for neural crest development and neural folding in Xenopus [61]. In the mouse TRAF4 deficiency can induce defects in neural tube closure [62]. This protein also participates in the control of myelination [62]. However, transcripts of two Traf genes (Traf1, Traf5) were not detected.
TRIM E3 Ub ligases
Proteins of the tripartite motif (TRIM) family are multi-functional proteins possessing a E3 Ub ligase activity. Absent in yeast, TRIM proteins are required for activation of the mammalian autophagy system and function also as critical regulators of innate immunity [63]. Several families and sub-families of TRIM proteins are identified (C-I to C-XI), plus a group of unclassified TRIM lacking a RING-finger domain [63]. More than eighty genes were analysed. Taken together, transcripts of thirty-two genes coding for RING-finger domain TRIM and only one TRIM without RING-finger domain were found (Fig. 5). Six Trim genes predominated: Trim28, Trim32, Trim35, Trim46, Trim59 and Trim67. The latter one was both the most expressed Trim gene and one of the most up-regulated gene analysed in the present report. No transcripts were detected before E13, and the TPM values increased from 12 (at E13) to 253 (at PN1). Overall, the transcript abundance increased by a factor 21 during embryonic development. The most significant elevation was noted between E13 and E17, indicating that TRIM67 is a ligase dispensable during neurogenesis but crucially essential for post-mitotic cells and the maturation of the cerebral cortex (Fig. 5). These data are in line with a previous report showing that TRIM67 proteins are highly expressed in the developing and mature brain, but not found in non-neuronal tissues [58]. Its protein expression peaked late embryonically and perinatally, indicating that it is involved in neuronal development after the proliferative period. Deletion of the Trim67 gene causes malformations in several brain regions associated to cognitive and behavioural impairments [58]. The molecular role of TRIM67 in brain development as well as the nature of its substrates are however largely ignored.
Trim35 expression was not as markedly regulated as Trim67 but it was nevertheless expressed at all ages except PN1. Its TPM values augmented from ⁓170 to 230 from E11 to PN1, reflecting a 35% augmentation of Trim35 transcripts abundance (Fig. 5). The third most prominent gene of this family was Trim28. Its expression was down-regulated with TPM values decreasing from ⁓240 to 120 between E11 to PN1. This repression occurred after E13, indicating that TRIM28 (KAP1 or TIF1b) exerts important roles during the proliferative period. TRIM28 is an epigenetic co-repressor protein highly expressed both in the developing and adult brain [59]. Its absence in mice is embryonically lethal (at around embryonic day 5.5). TRIM28 has been proposed to be a specific SUMO E3 ligase [63]. In murine and human brains, TRIM28 functions as a transcriptional regulator of neurodevelopmental gene programs important for brain development [59].
The other important Trim genes were Trim32, Trim46, and Trim59. The first two ones were up-regulated: their abundance of transcripts increased markedly after E13. For instance, the TPM values increased by a factor 2.6 and 8.7 for Trim32 and Trim46, respectively, between E11 and PN1 (Fig. 5). Trim46 was one of the most induced gene (⁓9-fold increased). Accumulation of TRIM32 proteins into neural cells favours their commitment to the neuronal lineage [60]. Following its translocation to the nucleus, TRIM32 targets c-Myc for proteasomal degradation which initiates neuronal differentiation [64]. Concerning Trim59, another highly regulated gene, its TPM values diminished by a factor ⁓9 (from 149 to 17 TPM) (Fig. 5). These changes in transcripts abundance occurred primarily after the peak of neurogenesis (E13). The mRNA levels were much highest during the proliferative periods of corticogenesis. TRIM59 proteins are abundantly expressed in some organs like spleen, stomach and ovary but they are also found at lower levels in brain, lung, kidney, muscle and intestine [48]. Again, it is interesting to note the high expression level of actors known to regulate carcinogenesis. This is for instance the case of TRIM28, TRIM32, and TRIM59 that are aberrantly overexpressed in some cancers. For instance, associated with proteins of the melanoma-associated antigen (MAGE) family, TRIM28 favours the progression of carcinogenesis via suppression of autophagy [63]. It is also worthy of note that many E3 Ub ligases such as MARCH and TRIM proteins known for their roles in immune responses are strongly expressed in the developing cerebral cortex.
B. Multi-subunits RING E3
Three families of multimeric RING E3 were considered in the present report: i) cullin RING ligases, ii) APC/C E3, and iii) Fanconi anemia complex.
B.1. Cullin RING Ligases (CRLs)
Cullin RING Ligases (CRLs) represent the largest family of E3 Ub ligases. They are complex molecular entities assembled from several independent subunits. CRLs (CRL1-9) comprise a cullin (Cul) scaffold associated to a RING-box protein and an adaptor protein. They also require a substrate recognition element that is an interchangeable subunit defining the target protein to be ubiquitinated. CRL3 is a notable exception to this rule where the same molecular entity (BTB or Broad complex, Tramtrack, Bric-a-brac) serves as both adaptor and substrate receptor [35]. The existence of several cullins, RING-box proteins, adaptor proteins, and hundreds of substrate recognition proteins contribute to generate a wide range of combinations giving rise to a multitude of functionally distinct CRLs [65]. Of note, the large repertoire of genes encoding substrate receptors was not analysed in this study. Table 2 gives an overview of the multi-subunit structure of CRLs and their modularity.
Table 2
gives an overview of the multi-subunit structure of CRLs and their modularity.
Type of CRL | Cullin scaffold | RING-finger protein | Adaptor protein | Substrate recognition protein |
CRL1 | CUL1 | ROC1 (Rbx1) | Skp1 | F-box |
CRL2 | CUL2 | ROC1 (Rbx1) | Elongin B / Elongin C | VHL-box |
CRL3 | CUL3 | ROC1 (Rbx1) | BTB |
CRL4A | CUL4A | ROC1 (Rbx1) | DDB1 | DCAF |
CRL4B | CUL4B | ROC1 (Rbx1) | DDB1 | DCAF |
CRL5 | CUL5 | ROC2 (Rbx2) | Elongin B / Elongin C | SOCS-box |
CRL7 | CUL7 | ROC1 (Rbx1) | Skp1 | Fbw8 |
CRL9 | CUL9 (PARC) | ? | ? | ? |
B.1.1 Cullin scaffold
Transcripts of nine cullin genes (Cul1-3, 4a, 4b, 5, 7, and Cul9 or Parc) were detected with Cul7 being the predominant member of this group. Its TPM values slightly decreased from 103 to 82 from E11 to PN1 (Fig. 6A). The CUL7 protein, only present in chordates, participates in the control of embryonic development. CUL7 knockout mice display neonatal lethality and mutations in the human Cul7 gene are linked to the growth retardation disorder 3-M syndrome [65]. The expression level of the human Cul7 gene is increased in glioblastoma tissues, compared to normal brain tissues. Furthermore, Cul7 facilitates the proliferation, invasion and migration of glioma cells by activating the NF-κB pathway [66]. This is line with the current view of CUL7 as an oncogene [67]. It is however interesting to note elevated levels of Cul7 expression in a healthy (non-tumorigenous) brain tissue. The neuronal functions of the scaffold protein CUL7 are scarcely understood. It is highly expressed in the developing rat brain [68] [69]. Only two F-box proteins are known to interact with CUL7: FBXW8 and FBXW11 [67]. Their genes were expressed but in an opposite manner: the abundance of Fbxw8 transcripts diminished from 16 to 10 TPM whereas the abundance of Fbxw11 transcripts increased from 12 to 19 TPM from E11 to PN1. CUL7 is, together with the F-box protein FBXW8, associated with the Golgi apparatus of neuronal cells and is required for the growth of dendrites (but not of axons) in neurons of the mammalian brain [68]. CUL7 is also found at synaptic sites, controlling the degradation of Eag1, a potassium channel of the plasma membrane participating in the regulation of membrane excitability [69]. Due to its high expression level and its synaptic localization, CUL7 could be an important modulator of neuronal excitability in the brain. Within the CRL7 E3 ligase complex the scaffold protein CUL7 is also associated with the adaptor protein Skp1 and the RING finger protein ROC1 that contains the E2 enzyme-binding domain. The expression of the Skp1 and Rbx1 genes will be discussed below.
The other Cul genes had low levels of TPM values (around 10–20 TPM). The expression of the minor Cul9 gene was developmentally regulated: its transcripts abundance augmented by a factor of ⁓eight between E11 and PN1 (from ⁓2 to 19 TPM) (Fig. 6A). It was one of the most upregulated gene analysed in this study.
B.1.2 Adaptor proteins
Adaptor proteins are attached to the cullin scaffold. The following four genes were considered: Skp1 (Skp1a), Elob (elongin B), Eloc (elongin C) and Ddb1. Three of them were highly expressed with TPM values ≥ 180: Skp1a, Elob, and Ddb1 (Fig. 6B). They displayed distinct patterns of expression: Elob was expressed at constant level, whereas Ddb1 expression was negatively regulated (Fig. 6B). The abundance of Ddb1 transcripts, the major gene of this subgroup, was reduced drastically during embryonic development, decreasing from 351 from 162 TPM (Fig. 6B).
B.1.3 RING finger proteins
The RING finger proteins present in E3 ligases function as docking sites for E2 enzymes. The TPM values of the Rbx1 and Rbx2 (Rnf7) genes were nearly identical at E11 (89 and 86, respectively) (Fig. 6C). They displayed differing expression patterns: the abundance of Rbx1 transcripts increased (with a peak at E17 with 109 TPM) whereas it decreased for Rbx2 (43 TPM at PN1). Overall, Rbx1 was the predominant RING finger protein encoding gene of the cerebral cortex during development (Fig. 6C).
B.2. Anaphase-promoting complex/cyclosome (APC/C) E3
E3 Ub ligases anaphase-promoting complex/cyclosome (APC/C) are well-known for their control of the cell cycle, regulating mitotic progression and exit. They are strongly expressed in post-mitotic neurons where they play a role in dendrite and axon arborisation, and also in synaptogenesis [9]. APC/C ligases are large multi-subunit complexes consisting of three sub-complexes: a catalytic core (APC2, APC10, APC11), a scaffolding platform (APC1, APC4, APC5, APC15), and a substrate recognition module (or tetratricopeptide repeat -TPR- lobe) (APC3, APC6, APC7, APC8, APC12, APC13, APC16). In addition, CDC20 and CDH1 are co-activators (also designed as substrate receptors) essential for the activity of the APC/C ligase [70] [71]. The expression of fourteen APC encoding genes (Anapc) and two co-activator encoding genes (Cdc20, Cdh1) were analysed.
Anapc2 (100–115 TPM) and Anapc11 (⁓49 TPM) were the major genes of the catalytic core. Transcripts of the other component (Anapc10) were near above the detection level (2–3 TPM) (Fig. 7A). With TPM values ranging from 128 to 107 from E11 to PN1, Anapc5 was the predominant gene of the scaffolding platform. Anapc1 and Anapc4 presented comparable low levels of expression (TPM values of 20–40) whereas the Anapc15 was even less expressed (TPM values of 6–12) (Fig. 7A). Anapc6 (Cdc16) and Anapc8 (Cdc23) were the most expressed genes of the substrate recognition module (Fig. 7A). Taken together, transcripts of this subgroup were poorly or moderately abundant with TPM values ranging from 12 to 64 TPM. As shown in Fig. 7A, genes of the APC/C subgroup displayed nearly constant levels of transcripts throughout cortical formation suggesting basic function in cell physiology. It is worthy of note the extremely elevated expression of Cdc20 gene at early stages of cortical development followed by a sharp diminution at E17. Its TPM values declined from ⁓350 − 310 at E11-E13 to 44 − 18 at E17-PN1. Overall, the abundance of Cdc20 transcripts diminished by a factor ⁓20, showing that this gene had a marked temporal pattern of expression. The high abundance of Cdc20 transcripts corresponded to periods of cell production (E11-E13) strongly suggesting a role of Cdc20 in cell proliferation. The expression of the other co-activator encoding gene Cdh1 (Fzr1) was not developmentally regulated. Constant levels of Cdh1 transcripts were found during embryonic development (TPM values of 89–90). Chd1 proteins are required for neurogenesis in vivo [72]. It seemed however that the regulation of the co-activator gene Cdc20 is a central determinant affecting the functionality of the APC/C E3 Ub ligases in the embryonic cerebral cortex. The APC/C E3 Ub ligases ubiquitinate their substrates in conjunction with a limited set of E2 Ub-conjugating enzymes: UBE2S, UBCH10 (UBE2C) and, to a lesser extent, UBCH5 (UBE2D1) [70] [71]. As shown in Fig. 1D, Ube2c was the most expressed of these three E2 enzymes. Of interest, Ube2c and Cdc20 displayed similar patterns of expression (Fig. 7B). The decline of Cdc20 expression mirrored the marked repression of the expression of Ube2c.
B.3. Fanconi anemia (FA) E3
The classification of the components of the Fanconi anemia (FA) complex was established according to [73] and the Fanconi anemia mutation database https://www2.rockefeller.edu/fanconi/). The following modules were considered: DNA anchor complex, core complex, the ubiquitinated ID2 complex (FANCI/FAND2), nucleases, DNA repair factor and other actors (Fig. 8). Altogether, twenty-seven FA genes were retained, including genes (Faap20, Faap24, Faap100) coding for regulatory subunits associated to the core complex. The expression of fourteen genes was repressed during embryonic development. Five genes were exclusively expressed during the neurogenesis period (E11-E13): Fanca, fancd2, Fanci, Fancn, and Fanco. Moreover, transcripts of four genes were found exclusively between E11 and E17 but not at PN1: Fancb, Fancr (Rad51), Fancs ( Brca1) and Fanct (Ube2t). Overall, these FA genes were extremely poorly expressed (TPM values < 10) (Fig. 8). The major genes were Fancf, Fancg, Fancv and Fancw, with TPM values ranging from 40 to 110 (Fig. 8). It is worthy of note that FANCL is the only protein of the FA complex displaying a ligase activity, described as a RING E3 ligase. However, no transcripts of its gene (Fancl) were found. Two other genes were also not expressed: Fancc and fancj (brip1). FANCC is, together with FANCL, a member of the core complex. These transcriptomic data suggest that FA E3 ligase was poorly or not active during embryonic development. Of note, Ube2T, the E2 working in concert with FA E3 ligases, was also very poorly expressed with TPM values diminishing from 17 to 4 between E11 to E17, and even no transcripts detected at PN1 (Fig. 1D). The FA complex is commonly described as a machinery recruited to DNA lesions and playing a role in DNA repair. Most of the FA genes were poorly expressed in the developing cerebral cortex, suggesting poor or no activity under physiological developmental conditions.
These enzymes form another prominent class of E3 Ub ligases. They are characterized by a peculiar protein domain named U-box and structurally related to the RING finger family [74] [75]. U-box E3 Ub ligases serve as scaffolds that recruit and colocalize both a Ub-charged E2 and the substrate. An interesting property of mammalian U-box E3 Ub proteins is their ability to interact with molecular chaperones or co-chaperones such as Hsp90, Hsp70, DnaJc7, EKN1, CRN, and VCP [76]. U-box E3 Ub ligases can be found as monomers (i.e. UBE4) or homodimers (CHIP, PRPF19) [77]. Some U-box E3 Ub proteins have been introduced as E4 enzymes due to their involvement in the assembly of poly-Ub chains on substrates that are first ubiquitinated by a non U-box E3 Ub enzyme.
Nine genes were analysed: Stub1 (Chip), Prpf19 (Prp19), Ube4a (Ufd2b), Ube4b (Ufd2a), Ppil2 (Cyc4), Ubox5 (Uip5), Wdsub1, Act1 (Traf3ip2) and Aff4 (Fig. 9). Except Act1, transcripts of all U-box E3 Ub encoding genes were found. No clear developmental regulation pattern was observed for U-box ligases except for Prpf19 (Prp19), the second most expressed U-box E3 Ub gene. Its TPM values decreased from 250 to 180 from E11 to PN1, a ⁓30% reduction in transcript abundance during corticogenesis. The functions of the Prpf19 (Prp19) gene product are unknown but it is an essential protein since mouse Prpf19 (Prp19) null mutants are lethal [78]. With TPM values of 300–350, the Stub1 (Chip) gene was the predominant gene of this group and also one of the most expressed E3 Ub ligase genes. This highlights its physiological relevance during brain formation and development. The U-box E3 Ub ligase CHIP is able to tag misfolded or damaged proteins for subsequent proteasomal degradation. Proteomics analysis identified hundreds of potential CHIP substrates in HEK 293 cells [79]. The very high level of Stub1 (Chip) expression underscores the physiological importance of CHIP during the protein quality control process and clearance of abnormal proteins throughout embryonic development.
The UFD2a protein (coded by Ube4b/Ufd2a) is highly abundant in some brain areas like the cerebrum and cerebellum of 8 weeks old C57Bl6 mice [80]. Furthermore, immuno-histochemical data indicate that, in the cerebral cortex, the UFD2a protein is localized mainly in the cytoplasm of neurons. [80] proposed that UFD2a contributes to the ubiquitination of specific substrates related to neuronal function. The high abundance of UFD2a proteins previously observed in the adult mouse brain is in sharp contrast with the low mRNA abundance of Ube4b (Ufd2a) transcripts in the embryonic brain.