Merlin/NF2 suppresses tumorigenesis by inhibiting the E3 ubiquitin ligase CRL4(DCAF1) in the nucleus.

Current models imply that the FERM domain protein Merlin, encoded by the tumor suppressor NF2, inhibits mitogenic signaling at or near the plasma membrane. Here, we show that the closed, growth-inhibitory form of Merlin accumulates in the nucleus, binds to the E3 ubiquitin ligase CRL4(DCAF1), and suppresses its activity. Depletion of DCAF1 blocks the promitogenic effect of inactivation of Merlin. Conversely, enforced expression of a Merlin-insensitive mutant of DCAF1 counteracts the antimitogenic effect of Merlin. Re-expression of Merlin and silencing of DCAF1 implement a similar, tumor-suppressive program of gene expression. Tumor-derived mutations invariably disrupt Merlin's ability to interact with or inhibit CRL4(DCAF1). Finally, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. We propose that Merlin suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4(DCAF1).

Role(s) of G3BPs in Human Pathogenesis.

Ras-GTPase-activating protein (SH3 domain)-binding proteins (G3BP) are RNA binding proteins that play a critical role in stress granule (SG) formation. SGs protect critical mRNAs from various environmental stress conditions by regulating mRNA stability and translation to maintain regulated gene expression. Recent evidence suggests that G3BPs can also regulate mRNA expression through interactions with RNA outside of SGs. G3BPs have been associated with a number of disease states, including cancer progression, invasion, metastasis, and viral infections, and may be useful as a cancer therapeutic target. This review summarizes the biology of G3BP including their structure, function, localization, role in cancer progression, virus replication, mRNA stability, and SG formation. We will also discuss the potential of G3BPs as a therapeutic target. SIGNIFICANCE STATEMENT: This review will discuss the molecular mechanism(s) and functional role(s) of Ras-GTPase-activating protein (SH3 domain)-binding proteins in the context of stress granule formation, interaction with viruses, stability of RNA, and tumorigenesis.

Clinical Effect Analysis of Spinal Cord Electrical Stimulator Implantation for Diabetic Foot.

OBJECTIVE: To investigate the clinical effect of spinal cord electrical stimulator implantation in the treatment of a diabetic foot (DF). MATERIALS AND METHODS: We recruited 19 patients with DF who were admitted to Shengjing Hospital of China Medical University between January 2018 and May 2020. All the patients were treated with spinal cord electrical stimulator implantation. Skin temperature, degree of pain, quality of life (QOL) score, limb (toe) preservation, and nerve conduction velocity of the patients were compared pre- and postoperatively. RESULTS: The diameter and peak velocity of multisegment arteries in the lower limbs had significantly increased post surgery. Foot skin temperature significantly increased in patients with good effect. The postoperative visual analog scale score of the patients was significantly lower than that noted preoperatively (p < 0.05). The conduction velocities of the lower limb sensory nerves (eg, superficial peroneal nerve and sural nerve) and motor nerves (eg, common peroneal nerve and tibial nerve) had improved post surgery. Moreover, patients' QOL score had significantly improved postoperatively (p < 0.05). The limb (toe) salvage rate was 94.74%. CONCLUSION: The implantation of a spinal cord electrical stimulator for treating DF can effectively relieve pain and other associated symptoms. Additionally, this device can promote nerve function recovery and lower limb blood supply and reduce the risk of toe amputation; therefore, it is clinically effective and should be considered in the treatment of DF.

Roles of the ubiquitin ligase CUL4B and ADP-ribosyltransferase TiPARP in TCDD-induced nuclear export and proteasomal degradation of the transcription factor AHR.

The aryl hydrocarbon receptor (AHR) is a transcription factor activated by exogenous halogenated polycyclic aromatic hydrocarbon compounds, including the environmental toxin TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and naturally occurring dietary and endogenous compounds. The activated AHR enhances transcription of specific genes including phase I and phase II metabolism enzymes and other targets genes such as the TCDD-inducible poly(ADP-ribose) polymerase (TiPARP). The regulation of AHR activation is a dynamic process: immediately after transcriptional activation of the AHR by TCDD, the AHR is exported from the nucleus to the cytoplasm where it is subjected to proteasomal degradation. However, the mechanisms regulating AHR degradation are not well understood. Here, we studied the role of two enzymes reported to enhance AHR breakdown: the cullin 4B (CUL4B)AHR complex, an E3 ubiquitin ligase that targets the AHR and other proteins for ubiquitination, and TiPARP, which targets proteins for ADP-ribosylation, a posttranslational modification that can increase susceptibility to degradation. Using a WT mouse embryonic fibroblast (MEF) cell line and an MEF cell line in which CUL4B has been deleted (MEFCul4b-null), we discovered that loss of CUL4B partially prevented AHR degradation after TCDD exposure, while knocking down TiPARP in MEFCul4b-null cells completely abolished AHR degradation upon TCDD treatment. Increased TCDD-activated AHR protein levels in MEFCul4b-null and MEFCul4b-null cells in which TiPARP was knocked down led to enhanced AHR transcriptional activity, indicating that CUL4B and TiPARP restrain AHR action. This study reveals a novel function of TiPARP in controlling TCDD-activated AHR nuclear export and subsequent proteasomal degradation.

CUL4high Lung Adenocarcinomas Are Dependent on the CUL4-p21 Ubiquitin Signaling for Proliferation and Survival.

Cullin (CUL) 4A and 4B ubiquitin ligases are often highly accumulated in human malignant neoplasms and are believed to possess oncogenic properties. However, the underlying mechanisms by which CUL4A and CUL4B promote pulmonary tumorigenesis remain largely elusive. This study reports that CUL4A and CUL4B are highly expressed in patients with non-small cell lung cancer (NSCLC), and their high expression is associated with disease progression, chemotherapy resistance, and poor survival in adenocarcinomas. Depletion of CUL4A (CUL4Ak/d) or CUL4B (CUL4Bk/d) leads to cell cycle arrest at G1 and loss of proliferation and viability of NSCLC cells in culture and in a lung cancer xenograft model, suggesting that CUL4A and 4B are oncoproteins required for tumor maintenance of certain NSCLCs. Mechanistically, increased accumulation of the cell cycle-dependent kinase inhibitor p21/Cip1/WAF1 was observed in lung cancer cells on CUL4 silencing. Knockdown of p21 rescued the G1 arrest of CUL4Ak/d or CUL4Bk/d NSCLC cells, and allowed proliferation to resume. These findings reveal that p21 is the primary downstream effector of lung adenocarcinoma dependence on CUL4, highlight the notion that not all substrates respond equally to abrogation of the CUL4 ubiquitin ligase in NSCLCs, and imply that CUL4Ahigh/CUL4Bhigh may serve as a prognostic marker and therapeutic target for patients with NSCLC.

A common intronic single nucleotide variant modifies PKD1 expression level.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in PKD1 and PKD2 (PKD1/2), has unexplained phenotypic variability likely affected by environmental and other genetic factors. Approximately 10% of individuals with ADPKD phenotype have no causal mutation detected, possibly due to unrecognized risk variants of PKD1/2. This study was designed to identify risk variants of PKD genes through population genetic analyses. We used Wright's F-statistics (Fst) to evaluate common single nucleotide variants (SNVs) potentially favored by positive natural selection in PKD1 from 1000 Genomes Project (1KG) and genotyped 388 subjects from the Rogosin Institute ADPKD Data Repository. The variants with >90th percentile Fst scores underwent further investigation by in silico analysis and molecular genetics analyses. We identified a deep intronic SNV, rs3874648G> A, located in a conserved binding site of the splicing regulator Tra2-ß in PKD1 intron 30. Reverse-transcription PCR (RT-PCR) of peripheral blood leukocytes (PBL) from an ADPKD patient homozygous for rs3874648-A identified an atypical PKD1 splice form. Functional analyses demonstrated that rs3874648-A allele increased Tra2-ß binding affinity and activated a cryptic acceptor splice-site, causing a frameshift that introduced a premature stop codon in mRNA, thereby decreasing PKD1 full-length transcript level. PKD1 transcript levels were lower in PBL from rs3874648-G/A carriers than in rs3874648-G/G homozygotes in a small cohort of normal individuals and patients with PKD2 inactivating mutations. Our findings indicate that rs3874648G > A is a PKD1 expression modifier attenuating PKD1 expression through Tra2-ß, while the derived G allele advantageously maintains PKD1 expression and is predominant in all subpopulations.

GC-MLP: Graph Convolution MLP for Point Cloud Analysis.

With the objective of addressing the problem of the fixed convolutional kernel of a standard convolution neural network and the isotropy of features making 3D point cloud data ineffective in feature learning, this paper proposes a point cloud processing method based on graph convolution multilayer perceptron, named GC-MLP. Unlike traditional local aggregation operations, the algorithm generates an adaptive kernel through the dynamic learning features of points, so that it can dynamically adapt to the structure of the object, i.e., the algorithm first adaptively assigns different weights to adjacent points according to the different relationships between the different points captured. Furthermore, local information interaction is then performed with the convolutional layers through a weight-sharing multilayer perceptron. Experimental results show that, under different task benchmark datasets (including ModelNet40 dataset, ShapeNet Part dataset, S3DIS dataset), our proposed algorithm achieves state-of-the-art for both point cloud classification and segmentation tasks.

CRL4 Ubiquitin Pathway and DNA Damage Response.

DNA damage occurs in a human cell at an average frequency of 10,000 incidences per day by means of external and internal culprits, damage that triggers sequential cellular responses and stalls the cell cycle while activating specific DNA repair pathways. Failure to remove DNA lesions would compromise genomic integrity, leading to human diseases such as cancer and premature aging. If DNA damage is extensive and cannot be repaired, cells undergo apoptosis. DNA damage response (DDR) often entails posttranslational modifications of key DNA repair and DNA damage checkpoint proteins, including phosphorylation and ubiquitination. Cullin-RING ligase 4 (CRL4) enzyme has been found to target multiple DDR proteins for ubiquitination. In this chapter, we will discuss key repair and checkpoint proteins that are subject to ubiquitin-dependent regulation by members of the CRL4 family during ultraviolet light (UV)-induced DNA damage.

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

Dysregulation of CUL4A and CUL4B Ubiquitin Ligases in Lung Cancer.

The Cullin-RING ubiquitin ligase 4 (CRL4) is implicated in controlling cell cycle, DNA damage repair, and checkpoint response based on studies employing cell lines and mouse models. CRL4 proteins, including CUL4A and CUL4B, are often highly accumulated in human malignancies. Elevated CRL4 attenuates DNA damage repair and increases genome instability that is believed to facilitate tumorigenesis. However, this has yet to be evaluated in human patients with cancer. In our study, 352 lung cancer and 62 normal lung specimens of Asian origin were constructed into tissue microarrays of four distinct lung cancer subtypes. Expression of CUL4A, CUL4B, and their substrates was detected by immunohistochemistry and analyzed statistically for their prognostic value and association with DNA damage response and genomic instability. Our results show that both CUL4A and CUL4B are overexpressed in the majority of lung carcinomas (PCUL4A <0.001 and PCUL4B <0.001) and significantly associated with tumor size (PCUL4A <0.001 and PCUL4B = 0.002), lymphatic invasion (PCUL4A = 0.004 and PCUL4B <0.001), metastasis (PCUL4A = 0.019 and PCUL4B = 0.006), and advanced TNM stage (PCUL4A <0.001 and PCUL4B <0.001), which parallels gene amplification and abnormal activation of the canonical WNT signaling. Moreover, overexpression of CUL4A, but not CUL4B, is significantly associated with tobacco smoking (p = 0.01) and is inversely correlated with XPC and P21, both of which are substrates of CUL4A (PCUL4A = 0.019 and PCUL4B = 0.006). Higher levels of CUL4A or CUL4B are significantly associated with the overall survival of patients (PCUL4A <0.001 and PCUL4B <0.001) and progression-free survival (PCUL4A <0.001 and PCUL4B = 0.001). Our findings revealed that CUL4A and CUL4B are differentially associated with etiologic factors for pulmonary malignancies and are independent prognostic markers for the survival of distinct lung cancer subtypes.

SETting the clock for histone H4 monomethylation.

In this and a previous issue of Molecular Cell, Oda et al. (2010), Abbas et al. (2010), and Centore et al. (2010) determined that the H4K20 histone methyltransferase PR-Set7/Set8 is posttranslationally regulated by the PCNA-dependent CRL4(Cdt2) ubiquitin ligase.

Cell Autonomous and Nonautonomous Function of CUL4B in Mouse Spermatogenesis.

CUL4B ubiquitin ligase belongs to the cullin-RING ubiquitin ligase family. Although sharing many sequence and structural similarities, CUL4B plays distinct roles in spermatogenesis from its homologous protein CUL4A. We previously reported that genetic ablation ofCul4ain mice led to male infertility because of aberrant meiotic progression. In the present study, we generated Cul4bgerm cell-specific conditional knock-out (Cul4b(Vasa)),as well asCul4bglobal knock-out (Cul4b(Sox2)) mouse, to investigate its roles in spermatogenesis. Germ cell-specific deletion of Cul4bled to male infertility, despite normal testicular morphology and comparable numbers of spermatozoa. Notably, significantly impaired sperm mobility caused by reduced mitochondrial activity and glycolysis level were observed in the majority of the mutant spermatozoa, manifested by low, if any, sperm ATP production. Furthermore,Cul4b(Vasa)spermatozoa exhibited defective arrangement of axonemal microtubules and flagella outer dense fibers. Our mass spectrometry analysis identified INSL6 as a novel CUL4B substrate in male germ cells, evidenced by its direct polyubiquination and degradation by CUL4B E3 ligase. Nevertheless,Cul4bglobal knock-out males lost their germ cells in an age-dependent manner, implying failure of maintaining the spermatogonial stem cell niche in somatic cells. Taken together, our results show that CUL4B is indispensable to spermatogenesis, and it functions cell autonomously in male germ cells to ensure spermatozoa motility, whereas it functions non-cell-autonomously in somatic cells to maintain spermatogonial stemness. Thus, CUL4B links two distinct spermatogenetic processes to a single E3 ligase, highlighting the significance of ubiquitin modification during spermatogenesis.