| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 99 === ABSTRACT
The most prevalent cause of mental retardation (MR) is the X-linked mental retardation (XLMR) caused by mutations at various loci of X chromosome. Molecular genetic studies demonstrated that truncating or missense mutations of cullin 4B (CUL4B) gene located on the Xq24-q25 locus were observed in male MR patients. CUL4B mutation is one of the most frequently mutated genes underlying XLMR. Up to now, molecular pathogenic mechanism of mutant CUL4B-induced MR has not been investigated in detail. CUL4B binds to ROC1 or ROC2, resulting in the formation of CUL4B-RING E3 ubiquitin ligase. CUL4B mRNA is highly expressed in the neurons of cerebral cortex. Abnormal neuronal development and function in the frontal lobe is involved in the pathogenesis of XLMR. It is very likely that XLMR mutations of CUL4B impair the function of CUL4B-RING E3 ligase and cause resulting mental retardation and other syndromes.
We tested the hypothesis that CUL4B mutation-induced neurotoxicity and XLMR results from a defective proteolysis and resulting toxic accumulation of cyclin E in affected neurons. To test this hypothesis, our results showed that in contrast to wild-type CUL4B, XLMR mutant (R572C), (V745A) or (R388X) CUL4B failed to promote ubiquitylation and degradation of cyclin E in HEK 293cell. Cyclin E protein expression was upregulated in cultured neocortical neurons infected with lentiviruses containing shRNA of CUL4B, and protein expression of cyclin E was significantly downregulated in neocortical neurons overexpressing wild-type CUL4B. Protein level of cyclin E was not significantly altered in cultured neocortical neurons expressing XLMR mutant CUL4B. These findings suggest that wild-type CUL4B has a neuroprotective effect by enhancing cyclin E degradation and that XLMR mutation impairs the ability of CUL4B to enhance cyclin E degradation and exert neuroprotective effect.
Our results using cultured neocortical neurons of mouse frontal lobe indicated that CUL4B knockdown by shRNA of CUL4B elevated protein level of TSC2 and impaired mTOR signaling pathway, which was indicated by reduced protein expression of phospho-mTOR (Ser2448), phosph-70S6K and phospho-4E-BP1(Thr37/46). Overexpression of wild-type CUL4B promoted the degradation of TSC2 and enhanced the activity of mTORC1 pathway. Immunoprecipitation assays showed that wild-type CUL4B stably expressed in HEK 293 cells enhanced polyubiquitylation of TSC2 and that XLMR mutant CUL4B exhibited an impaired ability to polyubiquitinate TSC2. Furthermore, in contrast with wild-type CUL4B, XLMR mutant CUL4B expressed in cultured neocortical neurons failed to enhance mTORC1 signaling pathway activity. Our results suggest that XLMR mutations impair CUL4B-RING E3 ligase-mediated degradation of TSC2 and cause resulting accumulation of TSC2 and inhibition of mTOR activity in the brain, leading to an impairment of neuronal development, synaptic plasticity and cognitive function in the frontal cortex.
In summary, the present study provides the evidence that wild-type CUL4B exerts a neuroprotective effect and promotes the activity of mTORC1 pathway by promoting polyubiquitylation and degradation of cyclin E and TSC2. In contrast to wild-type CUL4B, XLMR mutant (R572C), (V745A) or (R388X) CUL4B fails to enhance ubiquitination of cyclin E and TSC2. XLMR mutations cause the loss of CUL4B function and impairment of mTOR signaling, which could result in the malfunction of frontal cortex and mental retardation.
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