Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation (XLMR). However, the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood. We have generated mice with targeted disruption of Cul4b, and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues. Cul4b, but not its paralog Cul4a, is expressed at high levels in extra-embryonic tissues post implantation. Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21^Cip1/WAF and G2/M cell cycle arrest, which could be partially rescued by silencing of p21^Cip1/WAF. Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice. Therefore, while dispensable in the embryo proper, Cul4b performs an essential developmental role in the extra-embryonic tissues. Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.     

X-linked intellectual disability gene CUL4B targets Jab1/CSN5 for degradation and regulates bone morphogenetic protein signaling

Cullin 4B (CUL4B) is a scaffold protein involved in the assembly of cullin-RING ubiquitin ligase (E3) complexes. Contemporary reports have identified multiple mutations of CUL4B gene as being causally associated with X-linked intellectual disability (XLID). Identifying the specific protein substrates will help to better understand the physiological functions of CUL4B. The current study identified Jun activation domain-binding protein (Jab1/CSN5) in the COP9 signalosome (CSN) complex as a novel proteolytic target for the CUL4B ubiquitin ligase complex. The impaired degradation of Jab1 was observed in cells after RNAi-mediated CUL4B depletion. Integrity of DDB1-CUL4B-ROC1 was further demonstrated to be indispensable for the degradation of Jab1. In addition, the degradation of Jab1 is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of Jab1. Interestingly, CUL4B-silenced cells were shown to exhibit abnormal upregulation of bone morphogenetic protein (BMP) signaling. Furthermore, in vivo studies of embryonic fibroblasts in Cul4b-deficient mice demonstrated Jab1 accumulation and increased activation of the BMP signaling pathway. Together, the current findings demonstrate the CUL4B E3 ubiquitin ligase plays a key role in targeting Jab1 for degradation, potentially revealing a previously undocumented mechanism for regulation of the BMP signaling pathway involved with the CUL4B-based E3 complex. This observation may provide novel insights into the molecular mechanisms underlying CUL4B-associated XLID pathogenesis.     

DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.     

Characterization of Nuclear Localization Signal in the N Terminus of CUL4B and Its Essential Role in Cyclin E Degradation and Cell Cycle Progression

CUL4A and CUL4B, which are derived from the same ancestor, CUL4, encode scaffold proteins that organize cullin-RING ubiquitin ligase (E3) complexes. Recent genetic studies have shown that germ line mutation in CUL4B can cause mental retardation, short stature, and other abnormalities in humans. CUL4A was observed to be overexpressed in breast and hepatocellular cancers, although no germ line mutation in human CUL4A has been reported. Although CUL4A has been known to be involved in a number of cellular processes, including DNA repair and cell cycle regulation, little is known about whether CUL4B has similar functions. In this report, we tested the functional importance of CUL4B in cell proliferation and characterized the nuclear localization signal (NLS) that is essential for its function. We found that RNA interference silencing of CUL4B led to an inhibition of cell proliferation and a prolonged S phase, due to the overaccumulation of cyclin E, a substrate targeted by CUL4B for ubiquitination. We showed that, unlike CUL4A and other cullins that carry their NLS in their C termini, NLS in CUL4B is located in its N terminus, between amino acid 37 and 40, KKRK. This NLS could bind to importin α1, α3, and α5. NLS-deleted CUL4B was distributed in cytoplasm and failed to promote cell proliferation. Therefore, the nuclear localization of CUL4B mediated by NLS is critical for its normal function in cell proliferation.     

X-linked mental retardation gene CUL4B targets ubiquitylation of H3K4 methyltransferase component WDR5 and regulates neuronal gene expression

CUL4B, encoding a scaffold protein for the assembly of Cullin4B-Ring ubiquitin ligase (CRL4B) complexes, is frequently mutated in X-linked mental retardation (XLMR) patients. Here, we show that CUL4B, but not its paralog, CUL4A, targets WDR5, a core subunit of histone H3 lysine 4 (H3K4) methyltransferase complexes, for ubiquitylation and degradation in the nucleus. Knocking down CUL4B increases WDR5 and trimethylated H3K4 (H3K4me3) on the neuronal gene promoters and induces their expression. Furthermore, CUL4B depletion suppresses neurite outgrowth of PC12 neuroendocrine cells, which can be rescued by codepletion of WDR5. XLMR-linked mutations destabilize CUL4B and impair its ability to support neurite outgrowth of PC12 cells. Our results identify WDR5 as a critical substrate of CUL4B in regulating neuronal gene expression and suggest epigenetic change as a common pathogenic mechanism for CUL4B-associated XLMR.     

CUL4B ubiquitin ligase in mouse development: A model for human X-linked mental retardation syndrome?

CUL4B, a member of the cullin-RING ubiquitin ligase family, is frequently mutated in X-linked mental retardation (XLMR) patients. The study by Liu et al. showed that Cul4b plays an essential developmental role in the extra-embryonic tissues, while it is dispensable in the embryo proper during mouse embryogenesis. Viable Cul4b-null mice provide the first animal model to study neuronal and behavioral deficiencies seen in human CUL4B XLMR patients.CUL4 is a member of the cullin-RING ubiquitin ligase family, the largest E3 ligase family, which appears to account for ∼20% of total protein degradation by the ubiquitin-proteasome system^1,2,3. CUL4 is conserved during evolution from yeast to human. In yeast, CUL4 encodes a single gene, but mammalian cells express two closely related paralogs, CUL4A and CUL4B with about 82% sequence identity. CUL4A and CUL4B assemble structurally similar E3 complexes through binding to an adaptor protein (DDB1) and a substrate receptor protein (DCAF) at the N-terminus, and a RING protein RBX1 at the C-terminus (Figure 1), and share functional redundancy in targeting substrates such as p21 and Cdt1 for ubiquitination and degradation^1,2. The Cul4a-null mice are viable and display no abnormal development and growth phenotypes, likely due to functional compensation from Cul4b^4,5. The only phenotype associated with Cul4a abrogation is the reproductive defects seen with male but not female mice, resulting from differential non-overlapping expression patterns of the two Cul4 genes during male meiosis⁶. On the other hand, germline deletion of Cul4b resulted in embryonic lethality around E9.5⁷, indicating a unique function of Cul4b that cannot be compensated by Cul4a during embryogenesis.Open in a separate windowFigure 1Differential expression of Cul4a and Cul4b in the embryo proper and extra embryonic tissues determines their fate. Before implantation, both Cul4a and Cul4b are expressed in the blastocyst. Following implantation, Cul4a is expressed in the embryo proper, but not in extra-embryonic tissues. Upon Cul4b deletion, p21 accumulates in extra-embryonic tissues to induce G2/M arrest and eventually embryonic death due to degeneration of extra-embryonic tissues. Expression of Cul4a in embryo prevents p21 accumulation and subsequent embryonic death.Mental retardation (MR) affects approximately 1%-3% of the population and is about 30% more common in males than in females⁸, suggesting a causal relationship with gene mutations on the X chromosome. To date, mutations in about 100 genes have been identified in X-linked MR (XLMR), much more than those found on autosomes⁹. In 2007, two independent groups reported that mutations of CUL4B (Xq24) ubiquitin ligase gene are associated with XLMR^10,11. CUL4B-deficient patients display a syndrome of delayed puberty, moderate short stature, hypogonadism, relative macrocephaly, central obesity, fine intention tremor, brachydactyly, and large tongue^10,11. Similarly, the neuronal and developmental deficiencies found in XLMR patients with CUL4B mutations are not compensated by CUL4A. The studies of the molecular pathogenesis of human XLMR are lagging partly due to the lack of an animal model for the disease.In the most recent study published in Cell Research, Zhou and coworkers¹² attempted to generate conditional Cul4b knockout mice with targeted deletion of Cul4b at exons 4 and 5, giving rise to a non-functional Cul4b fragment lacking both the DDB1-binding domain and the cullin homology domain for RBX1 recruitment. The chicken-actin (CAG)-Cre was used, which drives Cre-mediated recombination at the early zygote stage, leading to Cul4b deletion in both the embryo proper and extra-embryonic tissues. Like human CUL4B, the mouse Cul4b is also located on the X-chromosome. Intercrossing of male CAG-Cre with female Cul4b^fl/+ revealed that hemizygous deletion of Cul4b causes embryonic lethality. No embryos with the genotype of Cul4b^−/y survived beyond E9.5. Interestingly, the heterozygous Cul4b^+/− embryos also die in the uterus before E13.5, suggesting that the paternal X chromosome undergoes imprinted inactivation with only trace amount, if any, of Cul4b expression remaining in extra-embryonic tissues. Detailed analysis of dissected embryos revealed that dying Cul4b^+/− embryos (E12.5) lack blood supply from the yolk sacs, whereas the Cul4b^−/y embryos (E8.5) showed remarkable reduction in proliferation with growth arrest at G2/M and enhanced apoptosis. The authors went on and investigated why Cul4a failed to compensate the loss of Cul4b, and found a dynamic expression pattern, differing between two forms, during early embryonic development. Prior to implantation, both Cul4 proteins are detectable in the blastocysts. Shortly after implantation, while both forms are expressed in the embryo proper, only Cul4b is expressed in the extra-embryonic tissues. Thus, upon Cul4b deletion, extra-embryonic tissues without Cul4a compensation degenerate, eventually leading to embryonic death. Consistently, when the authors deleted Cul4b in the epiblast using the Sox2-Cre (targeted Cul4b deletion in embryos proper only), viable Cul4b-null mice are produced likely due to Cul4a compensation. Thus, Cul4b is essential for the development of extra-embryonic tissues, but is dispensable for embryogenesis itself.To study the potential underlying mechanism(s) of embryonic lethality upon Cul4b deletion in extra-embryonic tissues, the authors used an extra-embryonic cell line (XEN). Cul4b knockdown induced a remarkable cell cycle arrest at the G2/M phase, consistent with observation made in Cul4b-null embryos, and robust accumulation of p21, a universal inhibitor of cyclin dependent kinase and a known substrate of Cul4¹. To determine whether accumulated p21 is responsible for the G2/M arrest, the authors simultaneously knocked down both Cul4b and p21 in XEN cells and observed a partial abrogation of growth arrest, suggesting that p21 plays a causal role, at least in part. Unfortunately, due to unavailability of anti-mouse p21 antibody specific for immunohistochemical staining, the authors were not able to show if p21 is indeed accumulated in extra-embryonic tissues upon Cul4b deletion. However, whether p21 indeed plays a causal role in embryonic death upon Cul4b deletion can be unequivocally determined by a rescuing experiment in which simultaneous deletion of p21 should abrogate or at least delay embryonic lethality, if it is causal. Nevertheless, the study by Zhou''s group can be summarized as follows. Before implantation, both Cul4a and Cul4b ubiquitin ligases are expressed in the blastocyst (inner cell mass and trophoblast cells). Following embryo implantation, while Cul4b is expressed in both the embryo proper and extra embryonic tissues, Cul4a is only expressed in the embryo proper. The CAG-Cre-driven Cul4b deletion (in both the embryo proper and extra-embryonic tissues) causes significant p21 accumulation in Cul4a non-expressing extra-embryonic tissues, resulting in G2/M arrest, followed by embryonic death due to degeneration of extra-embryonic tissues. On the embryo side, Cul4b deletion has no detrimental consequence, benefiting from the compensatory effect of Cul4a for p21 targeting. The same holds true when Cul4b is deleted driven by embryonic specific Sox2-Cre (Figure 1).It is noteworthy that the studies by Zhou''s group revealed two distinct differences between Cul4b KO mice and CUL4B-associated XLMR patients. First, Cul4b deletion at the zygote stage causes embryonic lethality, whereas XLMR patients with CUL4B mutations live to adulthood. Second, the Cul4b-null allele cannot be transmitted from the mother to the offspring, whereas human XLMR patients inherit X-linked CUL4B mutations from their mothers. Nevertheless, viable Cul4b-null mice (upon epiblast ablation by Sox2-Cre) provide the first mouse model for mechanistic study of human XLMR diseases associated with CUL4B mutations in the following three aspects:First, as noted earlier, human CUL4B XLMR patients have multiple neuronal and developmental defects. An obvious follow-up study will be to use this mouse model for neurological and behavioral analyses to determine whether Cul4b-null mice indeed present some of human XLMR symptoms.Second, this model can also be used to validate whether accumulation of Cul4b substrates during various stages of brain development indeed plays a pathogenic role and contributes to the clinical symptoms of XLMR patients. For instance, WDR5, a recently identified gene affecting general cognitive ability¹³, was found to be a novel nuclear substrate of CUL4B, but not CUL4A¹⁴. Investigation into whether WDR5 is abnormally accumulated upon Cul4b deletion in vivo would rule in or rule out its potential association with human XLMR, although it was not the case in this study using an extra-embryonic cell line in vitro.Third, the viability of Cul4b-null mice upon epiblast-specific deletion provides opportunities to study neuronal specific ablation of Cul4b in association with the pathogenesis of CUL4B-associated XLMR. For example, Cul4b is expressed at high levels in the hippocampus and cerebrum of mouse brains; both regions are affected in MR patients¹⁵. Thus, the use of Cre mouse lines that target the deletion of Cul4b in the entire brain, selected brain areas, or specific neuronal cells in both spatial and temporal manners¹⁶ would reveal potential contributions of particular regions and cell types to the development and symptoms of CUL4B-associated XLMR.A number of questions that warrant future investigation remain unanswered. First, in addition to p21, what are the other Cul4B substrates, which also contribute to degeneration of extra-embryonic tissues upon Cul4b deletion, since simultaneous deletion of p21 only partially rescues the growth defects? Second, besides the difference in tissue/cell specific expression seen in this study, are Cul4a and Cul4b targeting a unique set of substrates non-redundantly, thus differentiating their physiological functions? A related question will be why CUL4A cannot compensate for the loss of CUL4B in CUL4B-associated XLMR patients? Third, what is the pathogenic mechanism for CUL4B-associated XLMR? Is it mainly due to pathological accumulation of many CUL4B substrates? Answers to these questions may offer insights into potential therapeutic strategies for the treatment of CUL4B-associated XLMR patients.In summary, the findings reported by Zhou''s group provide the first convincing evidence that demonstrates an essential role of Cul4b in the development of extra-embryonic tissues during mouse embryogenesis. The viable Cul4b conditional knockout mice, generated in this study, may serve as the first mouse model for future mechanistic studies of neuronal and behavioral deficiencies of human XLMR associated with CUL4B mutations. We look forward to more exciting discoveries of how Cul4b deficiency leads to the development of XLMR in years to come.     

HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4^DCAF1). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4^DCAF1 and CRL4^DCAF1-Vpr E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4^DCAF1 E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.     

Deficiency for the Ubiquitin Ligase UBE3B in a Blepharophimosis-Ptosis-Intellectual-Disability Syndrome

Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.     

DCAF26, an Adaptor Protein of Cul4-Based E3, Is Essential for DNA Methylation in Neurospora crassa

DNA methylation is involved in gene silencing and genome stability in organisms from fungi to mammals. Genetic studies in Neurospora crassa previously showed that the CUL4-DDB1 E3 ubiquitin ligase regulates DNA methylation via histone H3K9 trimethylation. However, the substrate-specific adaptors of this ligase that are involved in the process were not known. Here, we show that, among the 16 DDB1- and Cul4-associated factors (DCAFs) encoded in the N. crassa genome, three interacted strongly with CUL4-DDB1 complexes. DNA methylation analyses of dcaf knockout mutants revealed that dcaf26 was required for all of the DNA methylation that we observed. In addition, histone H3K9 trimethylation was also eliminated in dcaf26^KO mutants. Based on the finding that DCAF26 associates with DDB1 and the histone methyltransferase DIM-5, we propose that DCAF26 protein is the major adaptor subunit of the Cul4-DDB1-DCAF26 complex, which recruits DIM-5 to DNA regions to initiate H3K9 trimethylation and DNA methylation in N. crassa.     

CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1

The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant’s resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1.     

Mutations in ZDHHC9, which encodes a palmitoyltransferase of NRAS and HRAS, cause X-linked mental retardation associated with a Marfanoid habitus

We have identified one frameshift mutation, one splice-site mutation, and two missense mutations in highly conserved residues in ZDHHC9 at Xq26.1 in 4 of 250 families with X-linked mental retardation (XLMR). In three of the families, the mental retardation phenotype is associated with a Marfanoid habitus, although none of the affected individuals meets the Ghent criteria for Marfan syndrome. ZDHHC9 is a palmitoyltransferase that catalyzes the posttranslational modification of NRAS and HRAS. The degree of palmitoylation determines the temporal and spatial location of these proteins in the plasma membrane and Golgi complex. The finding of mutations in ZDHHC9 suggests that alterations in the concentrations and cellular distribution of target proteins are sufficient to cause disease. This is the first XLMR gene to be reported that encodes a posttranslational modification enzyme, palmitoyltransferase. Furthermore, now that the first palmitoyltransferase that causes mental retardation has been identified, defects in other palmitoylation transferases become good candidates for causing other mental retardation syndromes.     

E3 ubiquitin ligase Cullin4B mediated polyubiquitination of p53 for its degradation

Controlled protein ubiquitination through E3 ubiquitin ligases and degradation via 26S proteasome machinery is required for orderly progression through cell cycle, chromatin remodeling, DNA repair, and development. Each cullin-dependent ubiquitin ligase (E3) complex can recruit various substrates for their degradation. Cullin 4A (CUL4A) and Cullin 4B (CUL4B) are members of cullin family proteins that mediate ubiquitin dependent proteolysis. Though, these two cul4 genes are functionally redundant, Cullin 4B is not a substitute for all the Cullin 4A functions. Published report has shown that CUL4A interacts with p53 and induces its decay. Although, CUL4A has been known to control several cellular processes, little is known about CUL4B functions. Therefore, in this study, we analyzed the role of CUL4B on p53 polyubiquitination. Our stable cell line and transient transfection studies show that CUL4B indeed interacts with p53 and induces its polyubiquitination. Importantly, both CUL4A and CUL4B overexpressing cells show almost equal levels of p53 polyubiquitination. Moreover, we observed an increased level of polyubiquitination on p53 in CUL4B overexpressing stable cell line upon treatment with siRNA specific for CUL4A indicating that CUL4B plays a vital role in p53 stability. In addition, we have observed the differential expression of CUL4B in various eukaryotic cell lines and mouse tissues suggesting the important role of CUL4B in various tissues. Together, these observations establish an important negative regulatory role of CUL4B on p53 stability.     

Mutation in CUL4B, which encodes a member of cullin-RING ubiquitin ligase complex, causes X-linked mental retardation

We reevaluated a previously reported family with an X-linked mental retardation syndrome and attempted to identify the underlying genetic defect. Screening of candidate genes in a 10-Mb region on Xq25 implicated CUL4B as the causative gene. CUL4B encodes a scaffold protein that organizes a cullin-RING (really interesting new gene) ubiquitin ligase (E3) complex in ubiquitylation. A base substitution, c.1564C-->T, converted a codon for arginine into a premature termination codon, p.R388X, and rendered the truncated peptide completely devoid of the C-terminal catalytic domain. The nonsense mutation also results in nonsense-mediated mRNA decay in patients. In peripheral leukocytes of obligate carriers, a strong selection against cells expressing the mutant allele results in an extremely skewed X-chromosome inactivation pattern. Our findings point to the functional significance of CUL4B in cognition and in other aspects of human development.     

XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons

Truncating or missense mutation of cullin 4B (CUL4B) is one of the most prevalent causes underlying X-linked intellectual disability (XLID). CUL4B-RING E3 ubiquitin ligase promotes ubiquitination and degradation of various proteins. Consistent with previous studies, overexpression of wild-type CUL4B in 293 cells enhanced ubiquitylation and degradation of TSC2 or cyclin E. The present study shows that XLID mutant (R388X), (R572C) or (V745A) CULB failed to promote ubiquitination and degradation of TSC2 or cyclin E. Adenoviruses-mediated expression of wild-type CUL4B decreased protein level of TSC2 or cyclin E in cultured neocortical neurons of frontal lobe. Furthermore, shRNA-mediated CUL4B knockdown caused an upregulation of TSC2 or cyclin E. XLID mutant (R388X), (R572C) or (V745A) CUL4B did not downregulate protein expression of TSC2 or cyclin E in neocortical neurons. By promoting TSC2 degradation, CUL4B could positively regulate mTOR activity in neocortical neurons of frontal cortex. Consistent with this hypothesis, CUL4B knockdown-induced upregulation of TSC2 in neocortical neurons resulted in a decreased protein level of active phospho-mTOR^Ser2448 and a reduced expression of active phospho-p70S6K^Thr389 and phospho-4E-BP1^Thr37/46, two main substrates of mTOR-mediated phosphorylation. Wild-type CUL4B also increased protein level of active phospho-mTOR^Ser2448, phospho-p70S6K^Thr389 or phospho-4E-BP1^Thr37/46. XLID CUL4B mutants did not affect protein level of active phospho-mTOR^Ser2448, phospho-p70S6K^Thr389 or phospho-4E-BP1^Thr37/46. Our results suggest that XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons.     

Cullin 4B protein ubiquitin ligase targets peroxiredoxin III for degradation

Cullin 4B (CUL4B) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes. Recent studies have revealed that germ-line mutations in CUL4B can cause mental retardation, short stature, and many other abnormalities in humans. Identifying specific CUL4B substrates will help to better understand the physiological functions of CUL4B. Here, we report the identification of peroxiredoxin III (PrxIII) as a novel substrate of the CUL4B ubiquitin ligase complex. Two-dimensional gel electrophoresis coupled with mass spectrometry showed that PrxIII was among the proteins up-regulated in cells after RNAi-mediated CUL4B depletion. The impaired degradation of PrxIII observed in CUL4B knockdown cells was confirmed by Western blot. We further demonstrated that DDB1 and ROC1 in the DDB1-CUL4B-ROC1 complex are also indispensable for the proteolysis of PrxIII. In addition, the degradation of PrxIII is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of PrxIII. Furthermore, we observed a significant decrease in cellular reactive oxygen species (ROS) production in CUL4B-silenced cells, which was associated with increased resistance to hypoxia and H(2)O(2)-induced apoptosis. These findings are discussed with regard to the known function of PrxIII as a ROS scavenger and the high endogenous ROS levels required for neural stem cell proliferation. Together, our study has identified a specific target substrate of CUL4B ubiquitin ligase that may have significant implications for the pathogenesis observed in patients with mutations in CUL4B.     

Alternative Reading Frame Supports An Alternative Model for Retinoblastoma

The ubiqutin-proteasome system is the major pathway by which cells target proteins for degradation in a specific manner. The E3 ubiquitin ligase, which brings targeted proteins (substrates) and activated ubiquitin in close proximity, enabling covalent conjugation of ubiquitin to the substrate, is an essential component of this system. Of the E3 ligases, the cullin (CUL) ligases are of high interest because of their capacity to form multiple distinct E3 complexes to ubiquitinate a potentially large number of substrates. Of the six closely related cullins, very little is known about how specific substrates are recruited to CUL4-dependent ligases. A recent paper in Nature Cell Biology may shed some light on this issue as well as on the function of DDB1, a damaged-DNA binding protein that has long been associated with DNA repair.