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De novo variants in CUL3 are associated with global developmental delays with or without infantile spasms

Nakashima M, Kato M, Matsukura M, Kira R, Ngu LH, Lichtenbelt KD, et al.

J Hum Genet, 2020 Sep;65(9):727-734.
PMID: 32341456 DOI: 10.1038/s10038-020-0758-2

The ubiquitin-proteasome system is the principal system for protein degradation mediated by ubiquitination and is involved in various cellular processes. Cullin-RING ligases (CRL) are one class of E3 ubiquitin ligases that mediate polyubiquitination of specific target proteins, leading to decomposition of the substrate. Cullin 3 (CUL3) is a member of the Cullin family proteins, which act as scaffolds of CRL. Here we describe three cases of global developmental delays, with or without epilepsy, who had de novo CUL3 variants. One missense variant c.854T>C, p.(Val285Ala) and two frameshift variants c.137delG, p.(Arg46Leufs*32) and c.1239del, p.(Asp413Glufs*42) were identified by whole-exome sequencing. The Val285 residue located in the Cullin N-terminal domain and p.Val285Ala CUL3 mutant showed significantly weaker interactions to the BTB domain proteins than wild-type CUL3. Our findings suggest that de novo CUL3 variants may cause structural instability of the CRL complex and impairment of the ubiquitin-proteasome system, leading to diverse neuropsychiatric disorders.

Matched MeSH terms: Cullin Proteins/genetics*; Cullin Proteins/metabolism*
Fulltext Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex

Yuniati L, Lauriola A, Gerritsen M, Abreu S, Ni E, Tesoriero C, et al.

Cell Rep, 2020 05 19;31(7):107664.
PMID: 32433973 DOI: 10.1016/j.celrep.2020.107664

Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.

Matched MeSH terms: Cullin Proteins/metabolism*
Peg-4000 increased the mating efficiency of yeast-two hybrid screening process using pmf-box1 as bait

Nur Athirah Abd Hamid, Ismanizan Ismail

Sains Malaysiana, 2018;47:2961-2968.

Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by
the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box
protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS.
A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat
(FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system
(Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins.
Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a
bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA)
and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before
the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter
genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared
to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands
of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique.
Those genes may code for potential interacting proteins that needs further identification and confirmation.

Matched MeSH terms: Cullin Proteins
Arabidopsis at2g02870 loss of function mutants lead to enhanced production of hydroperoxide lyase pathway genes and products

Muhammad Naeem-ul-Hassan, Zamri Zainal, Ismanizan Ismail, Nur Athirah Abd Hamid, Muhammad Sajad

Sains Malaysiana, 2018;47:3003-3008.

F-box proteins containing variable C-terminal domains make an essential part of SKP1-Cullin-Ring box-F box (SCF)
complex. SCF complex catalyzes the final step to link the ubiquitin tag with the target protein, destined for degradation,
through F-box protein that confer overall substrate specificity to the complex. In this study, we analyzed the role of
At2g02870, a Kelch containing F-box protein from Arabidopsis thaliana, by using reverse genetics strategy. At2g02870
loss of function mutant lines (at2g02870) were analyzed and compared with wild type plants for the expression of genes
and products of hydroperoxide lyase (HPL) branch of oxylipin pathway. We found that the at2g02870 plants have enhanced
expression of HPL pathway genes and produce more green leaf volatiles (GLV) than the wild type plants. Our results
suggested that the gene is involved in the regulation of HPL pathway, possibly through the degradation of enzymes or/
and the regulatory factors of the pathway.

Matched MeSH terms: Cullin Proteins
Fulltext Inhibition of NEDD8 and FAT10 ligase activities through the degrading enzyme NEDD8 ultimate buster 1: A potential anticancer approach

Tan KL, Pezzella F

Oncol Lett, 2016 Dec;12(6):4287-4296.
PMID: 28101194 DOI: 10.3892/ol.2016.5232

The capabilities of tumour cells to survive through deregulated cell cycles and evade apoptosis are hallmarks of cancer. The ubiquitin-like proteins (UBL) proteasome system is important in regulating cell cycles via signaling proteins. Deregulation of the proteasomal system can lead to uncontrolled cell proliferation. The Skp, Cullin, F-box containing complex (SCF complex) is the predominant E3 ubiquitin ligase, and has diverse substrates. The ubiquitin ligase activity of the SCF complexes requires the conjugation of neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to cullin proteins. A tumour suppressor and degrading enzyme named NEDD8 ultimate buster 1 (NUB1) is able to recruit HLA-F-adjacent transcript 10 (FAT10)- and NEDD8-conjugated proteins for proteasomal degradation. Ubiquitination is associated with neddylation and FAT10ylation. Although validating the targets of UBLs, including ubiquitin, NEDD8 and FAT10, is challenging, understanding the biological significance of such substrates is an exciting research prospect. This present review discusses the interplay of these UBLs, as well as highlighting their inhibition through NUB1. Knowledge of the mechanisms by which NUB1 is able to downregulate the ubiquitin cascade via NEDD8 conjugation and the FAT10 pathway is essential. This will provide insights into potential cancer therapy that could be used to selectively suppress cancer growth.

Matched MeSH terms: Cullin Proteins
Fulltext SEMA3B but Not CUL1 as Marker for Pre-Eclampsia Progression

Samara TD, Liem IK, Prijanti AR, Andrijono

Malays J Med Sci, 2019 Jan;26(1):66-72.
PMID: 30914894 DOI: 10.21315/mjms2019.26.1.6

Background: An imbalance between pro- and anti-angiogenic factors contributes to impaired trophoblast invasion during pregnancy, leading to failure of uterine spiral artery remodeling, blood vessel ischemia, and pre-eclampsia (PE). Anti-angiogenic semaphorin 3B (SEMA3B) and pro-angiogenic cullin 1 (CUL1) are expressed in both the placenta and maternal blood. The present study investigated correlations between serum and placental SEMA3B as well as CUL1 levels in late-onset PE.
Methods: This cross-sectional study included 50 patients with late-onset (≥ 32 weeks gestation) PE. Maternal serum was obtained before delivery, and placentas were obtained immediately after delivery. SEMA3B and CUL1 levels were evaluated by ELISA. Results were statistically analysed by Spearman correlation test, with a P < 0.05 considered statistically significant.
Results: While elevated serum SEMA3B levels significantly correlated with increased placental SEMA3B levels in late-onset PE (R = 0.620, P = 0.000), alteration of serum CUL1 levels did not correlate with alteration of placental CUL1.
Conclusion: Alteration of circulating maternal SEMA3B, but not CUL1, levels can potentially be used to monitor PE progression during pregnancy.

Matched MeSH terms: Cullin Proteins