Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism.

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.

Distinct nuclear and cytoplasmic assemblies and interactomes of the mammalian CTLH E3 ligase complex.

The C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit E3 ubiquitin ligase and its cellular functions are poorly characterized. Although some CTLH subunits have been found to localize in both the nucleus and cytoplasm of mammalian cells, differences between the compartment-specific complexes have not been explored. Here, we show that the CTLH complex forms different molecular mass complexes in nuclear and cytoplasmic fractions. Loss of WDR26 severely decreased nuclear CTLH complex subunit levels and impaired higher-order CTLH complex formation, revealing WDR26 as a critical determinant of the nuclear stability of the CTLH complex. Through affinity purification coupled to mass spectrometry of endogenous RanBPM (also called RANBP9), a CTLH complex member, from nuclear and cytoplasmic fractions, we identified over 170 compartment-specific interactors involved in various conserved biological processes, such as ribonucleoprotein biogenesis and chromatin assembly. We validated the nuclear-specific RanBPM interaction with macroH2A1 and the cytoplasm-specific interaction with tankyrase-1/2 (encoded by TNKS and TNKS2). Overall, this study provides critical insights into CTLH complex function and composition in both the cytoplasm and nucleus.

WD-40 repeat protein 26 protects against oxidative stress-induced injury in astrocytes via Nrf2/HO-1 pathways.

BACKGROUND: Stroke is the leading cause of disability and the third leading cause of death in the world, and no effective treatment has been developed. Oxidative stress-induced cell injury and genomic instability is implicated in the pathogenesis of stroke, whose prognosis remains poor. METHODS: A model of cerebral ischemic/reperfusion injury model was established through four artery occlusions. This study was carried out using western blot, flow cytometry and RT-PCR on cell line U251-MG. The cytotoxic effect of H2O2 and expression of LDH, caspase-3, MDA and SOD was analyzed by assay kit. RESULTS: We found that the expression of WDR26 was induced in cerebral ischemia-reperfusion injury in vivo and the expression of WDR26 was induced by H2O2 in a dose- and time-dependent manner in vitro. WDR26 over-expression significantly suppressed H2O2-induced cell death and caspase-3-mediated apoptosis in U251-MG cells. In contrast, inhibition of WDR26 markedly enhanced cell death in U251-MG cells. In addition, WDR26 regulated oxidative stress response and induced Nrf2/HO-1 pathway. CONCLUSIONS: These findings suggest that WDR26 mediates H2O2-induced oxidative stress and cell injury, possibly by reducing the intrinsic apoptotic pathway and activating Nrf2 and HO-1 in astrocytes.

Structural and Functional Insights into GID/CTLH E3 Ligase Complexes.

Multi-subunit E3 ligases facilitate ubiquitin transfer by coordinating various substrate receptor subunits with a single catalytic center. Small molecules inducing targeted protein degradation have exploited such complexes, proving successful as therapeutics against previously undruggable targets. The C-terminal to LisH (CTLH) complex, also called the glucose-induced degradation deficient (GID) complex, is a multi-subunit E3 ligase complex highly conserved from Saccharomyces cerevisiae to humans, with roles in fundamental pathways controlling homeostasis and development in several species. However, we are only beginning to understand its mechanistic basis. Here, we review the literature of the CTLH complex from all organisms and place previous findings on individual subunits into context with recent breakthroughs on its structure and function.

Skraban-Deardorff syndrome: Six new cases of WDR26-related disease and expansion of the clinical phenotype.

Skraban-Deardorff syndrome (a disease related to variations in the WDR26 gene; OMIM #617616) was first described in a cohort of 15 individuals in 2017. The syndrome comprises intellectual deficiency, severe speech impairment, ataxic gait, seizures, mild hypotonia with feeding difficulties during infancy, and dysmorphic features. Here, we report on six novel heterozygous de novo pathogenic variants in WDR26 in six probands. The patients' phenotypes were consistent with original publication. One patient displayed marked hypotonia with an abnormal muscle biopsy; this finding warrants further investigation. Gait must be closely monitored, in order to highlight any musculoskeletal or neurological abnormalities and prompt further examinations. Speech therapy and alternative communication methods should be initiated early in the clinical follow-up, in order to improve language and oral eating and drinking.

Expanding the clinical phenotype of the ultra-rare Skraban-Deardorff syndrome: Two novel individuals with WDR26 loss-of-function variants and a literature review.

De novo variants in the WDR26 gene leading to haploinsufficiency have recently been associated with Skraban-Deardorff syndrome. This condition is an ultra-rare autosomal dominant neurodevelopmental disorder characterized by a broad range of clinical signs, including intellectual disability (ID), developmental delay (DD), seizures, abnormal facial features, feeding difficulties, and minor skeletal anomalies. Currently, 18 cases have been reported in the literature and for only 15 of them a clinical description is available. Here, we describe a child with Skraban-Deardorff syndrome associated with the WDR26 pathogenic de novo variant NM_025160.6:c.69dupC, p.(Gly24ArgfsTer48), and an adult associated with the pathogenic de novo variant c.1076G > A, p.(Trp359Ter). The adult patient was a 29-year-old female with detailed information on clinical history and pharmacological treatments since birth, providing an opportunity to map disease progression and patient management. By comparing our cases with published reports of Skraban-Deardorff syndrome, we provide a genetic and clinical summary of this ultrarare condition, describe the clinical management from childhood to adult age, and further expand on the clinical phenotype.

WDR26 Haploinsufficiency Causes a Recognizable Syndrome of Intellectual Disability, Seizures, Abnormal Gait, and Distinctive Facial Features.

We report 15 individuals with de novo pathogenic variants in WDR26. Eleven of the individuals carry loss-of-function mutations, and four harbor missense substitutions. These 15 individuals comprise ten females and five males, and all have intellectual disability with delayed speech, a history of febrile and/or non-febrile seizures, and a wide-based, spastic, and/or stiff-legged gait. These subjects share a set of common facial features that include a prominent maxilla and upper lip that readily reveal the upper gingiva, widely spaced teeth, and a broad nasal tip. Together, these features comprise a recognizable facial phenotype. We compared these features with those of chromosome 1q41q42 microdeletion syndrome, which typically contains WDR26, and noted that clinical features are consistent between the two subsets, suggesting that haploinsufficiency of WDR26 contributes to the pathology of 1q41q42 microdeletion syndrome. Consistent with this, WDR26 loss-of-function single-nucleotide mutations identified in these subjects lead to nonsense-mediated decay with subsequent reduction of RNA expression and protein levels. We derived a structural model of WDR26 and note that missense variants identified in these individuals localize to highly conserved residues of this WD-40-repeat-containing protein. Given that WDR26 mutations have been identified in ∼1 in 2,000 of subjects in our clinical cohorts and that WDR26 might be poorly annotated in exome variant-interpretation pipelines, we would anticipate that this disorder could be more common than currently appreciated.

A novel FBXO28 frameshift mutation in a child with developmental delay, dysmorphic features, and intractable epilepsy: A second gene that may contribute to the 1q41-q42 deletion phenotype.

Chromosome 1q41-q42 deletions have recently been associated with a recognizable neurodevelopmental syndrome of early childhood (OMIM 612530). Within this group, a predominant phenotype of developmental delay (DD), intellectual disability (ID), epilepsy, distinct dysmorphology, and brain anomalies on magnetic resonance imaging/computed tomography has emerged. Previous reports of patients with de novo deletions at 1q41-q42 have led to the identification of an evolving smallest region of overlap which has included several potentially causal genes including DISP1, TP53BP2, and FBXO28. In a recent report, a cohort of patients with de novo mutations in WDR26 was described that shared many of the clinical features originally described in the 1q41-q42 microdeletion syndrome (MDS). Here, we describe a novel germline FBXO28 frameshift mutation in a 3-year-old girl with intractable epilepsy, ID, DD, and other features which overlap those of the 1q41-q42 MDS. Through a familial whole-exome sequencing study, we identified a de novo FBXO28 c.972_973delACinsG (p.Arg325GlufsX3) frameshift mutation in the proband. The frameshift and resulting premature nonsense mutation have not been reported in any genomic database. This child does not have a large 1q41-q42 deletion, nor does she harbor a WDR26 mutation. Our case joins a previously reported patient also in whom FBXO28 was affected but WDR26 was not. These findings support the idea that FBXO28 is a monogenic disease gene and contributes to the complex neurodevelopmental phenotype of the 1q41-q42 gene deletion syndrome.

WDR26 promotes mitophagy of cardiomyocytes induced by hypoxia through Parkin translocation.

Myocardial ischemia is a heart condition caused by reduction of blood flow to the heart, preventing heart from receiving enough oxygen. Myocardial ischemia is the most common cause of death globally. Heart ischemic preconditioning (IPC) has a protective effect against myocardial cell death induced by ischemia and ischemia-reperfusion injury. WDR26 has recently been identified as a protein that is increased following rat cardiac IPC. WDR26 can promote the proliferation of H9c2 cells and protect cardiomyocytes against oxidative stress through inhibiting apoptosis. However, its role in myocardial ischemia is unclear. The aim of this study was to explore the role of WDR26 in myocardial ischemia and H9c2 cell hypoxia. Our results showed that WDR26 is induced by myocardial ischemia and H9c2 cell hypoxia. WDR26 protects H9c2 cells against hypoxia injury through inhibiting LDH release and increasing cell viability. WDR26 promotes hypoxia-induced autophagy in hypoxia of H9c2 cells. We further demonstrated that in H9c2 cell hypoxia, WDR26 increases mitochondrial membrane potential, thereby increases Parkin translocation of mitochondria. After Parkin is translocated at mitochondria, WDR26 can increase mitochondrial protein ubiquitination in hypoxia of H9c2 cells. WDR26 is a mediator of response to hypoxia, and WDR26 plays an important role in hypoxia-mediated autophagy and mitophagy. This study provides novel insights into the protective role of WDR26 in cardiomyocyte injury during hypoxia. WDR26 may serve as a potential target for the treatment of myocardial ischemia.

WDR26 functions as a scaffolding protein to promote Gßγ-mediated phospholipase C ß2 (PLCß2) activation in leukocytes.

We have recently identified WDR26 as a novel WD40 repeat protein that binds Gßγ and promotes Gßγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gßγ-mediated phospholipase C ß2 (PLCß2) activation in leukocytes. We show that WDR26 not only directly bound Gßγ but also PLCß2. The binding sites of WDR26 and PLCß2 on Gß1γ2 were overlapping but not identical. WDR26 used the same domains for binding Gßγ and PLCß but still formed a signaling complex with Gßγ and PLCß2 probably due to the fact that WDR26 formed a higher order oligomer through its Lis homology and C-terminal to LisH (LisH-CTLH) and WD40 domains. Additional studies indicated that the formation of higher order oligomers was required for WDR26 to promote PLCß2 interaction with and activation by Gßγ. Moreover, WDR26 was required for PLCß2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCß2 was sufficient to cause partial activation of PLCß2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCß2 membrane translocation and interaction with Gßγ, thereby enhancing PLCß2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gßγ signaling through a scaffolding protein.

Skraban-Deardorff intellectual disability syndrome-associated mutations in WDR26 impair CTLH E3 complex assembly.

Patients with Skraban-Deardorff syndrome (SKDEAS), a neurodevelopmental syndrome associated with a spectrum of developmental and intellectual delays and disabilities, harbor diverse mutations in WDR26, encoding a subunit of the multiprotein CTLH E3 ubiquitin ligase complex. Structural studies revealed that homodimers of WDR26 bridge two core-CTLH E3 complexes to generate giant, hollow oval-shaped supramolecular CTLH E3 assemblies. Additionally, WDR26 mediates CTLH E3 complex binding to subunit YPEL5 and functions as substrate receptor for the transcriptional repressor HBP1. Here, we mapped SKDEAS-associated mutations on a WDR26 structural model and tested their functionality in complementation studies using genetically engineered human cells lacking CTLH E3 supramolecular assemblies. Despite the diversity of mutations, 15 of 16 tested mutants impaired at least one CTLH E3 complex function contributing to complex assembly and interactions, thus providing first mechanistic insights into SKDEAS pathology.

Two Novel Variants of WDR26 in Chinese Patients with Intellectual Disability.

Skraban-Deardorff syndrome is a rare autosomal dominant genetic disease caused by variants in the WDR26 gene. Here, we report two Chinese patients diagnosed with Skraban-Deardorff syndrome caused by novel de novo, heterozygous pathogenic WDR26 variants c.977delA (p. 12 N326Ifs*2) and c.1020-2A>G (p. R340Sfs*29). Their clinical features were characterized by intellectual disability (ID), developmental delay, abnormal facial features and the absence of early-onset seizure, which expands the phenotype spectrum associated with Skraban-Deardorff syndrome. By comparing our cases with current reported cases of WDR26-related intellectual disability, we suggest that developmental delay, particularly in speech, and facial features including rounded palpebral fissures, depressed nasal root, full nasal tip and abnormal gums, represent the prominent clinical phenotypes for diagnosis of Skraban-Deardorff syndrome. Together, WDR26 variants and 1q41q42 deletions should feature prominently on the differential diagnosis of ID with distinctive facial features.

A novel long noncoding RNA lncWDR26 suppresses the growth and metastasis of hepatocellular carcinoma cells through interaction with SIX3.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Long noncoding RNAs (lncRNAs) are involved in the tumorigenesis and progression of some cancers. However, only a handful of lncRNAs have been functionally identified in HCC. In the present study, we identified a novel functional lncRNA in HCC, termed lncWDR26 (GenBank Accession no. RP11-365O16). Here, we reported that lncWDR26 was significantly downregulated in HCC tissues and cells. Moreover, decreased lncWDR26 expression correlates with larger tumor size, higher clinical stage, and tumor metastasis, and also predicts poor prognosis in patients with HCC. In HCC cells, overexpression of lncWDR26 inhibited growth and metastasis, both in vitro and in vivo. Mechanistically, lncWDR26 suppressed HCC growth and metastasis by inhibiting WDR26 transcription. Notably, lncWDR26 was associated with SIX homeobox 3 (SIX3), and this association was required for the repression of WDR26 transcription. Together, these results indicate that lncWDR26 is a tumor suppressor lncRNA that promotes tumor progression, leading us to propose that lncRNAs may serve as key regulatory hubs in HCC progression.

WDR26 is a new partner of Axin1 in the canonical Wnt signaling pathway.

The stability of ß-catenin is very important for canonical Wnt signaling. A protein complex including Axin/APC/GSK3ß phosphorylates ß-catenin to be degraded by ubiquitination with ß-TrCP. In the recent study, we isolated WDR26, a protein that binds to Axin. Here, we found that WDR26 is a negative regulator of the canonical Wnt signaling pathway, and that WDR26 affected ß-catenin levels. In addition, WDR26/Axin binding is involved in the ubiquitination of ß-catenin. These results suggest that WDR26 plays a negative role in ß-catenin degradation in the Wnt signaling pathway.

Upregulated WDR26 serves as a scaffold to coordinate PI3K/ AKT pathway-driven breast cancer cell growth, migration, and invasion.

The phosphatidylinositol 3-kinase (PI3K)/AKT pathway transmits signals downstream of receptor tyrosine kinases and G protein-coupled receptors (GPCRs), and is one of the most dysregulated pathways in breast cancer. PI3Ks and AKTs consist of multiple isoforms that play distinct and even opposite roles in breast cancer cell growth and metastasis. However, it remains unknown how the activities of various PI3K and AKT isoforms are coordinated during breast cancer progression. Previously, we showed WDR26 is a novel WD40 protein that binds Gßγ and promotes Gßγ signaling. Here, we demonstrate that WDR26 is overexpressed in highly malignant breast tumor cell lines and human breast cancer samples, and that WDR26 overexpression correlates with shortened survival of breast cancer patients. In highly malignant cell lines (MDA-MB231, DU4475 and BT549), downregulation of WDR26 expression selectively alleviated GPCR- but not EGF receptor-stimulated PI3K/AKT signaling and tumor cell growth, migration and invasion. In contrast, in a less malignant cell line (MCF7), WDR26 overexpression had the opposite effect. Additional studies indicate that downstream of GPCR stimulation, WDR26 serves as a scaffold that fosters assembly of a specific signaling complex consisting of Gßγ, PI3Kß and AKT2. In an orthotopic xenograft mouse model of breast cancer, disrupting formation of this complex, by overexpressing WDR26 mutants in MDA-MB231 cells, abrogated PI3K/AKT activation and tumor cell growth and metastasis. Together, our results identify a novel mechanism regulating GPCR-dependent activation of the PI3K/AKT signaling axis in breast tumor cells, and pinpoint WDR26 as a potential therapeutic target for breast cancer.